ROBO2 inhibitory compositions comprising SLIT2-binding extracellular domain of ROBO2

ABSTRACT

Provided herein are methods for the treatment of chronic kidney disease and proteinuria and for the diagnosis of chronic kidney disease and monitoring the effects of treatment on the progression of chronic kidney disease and proteinuria based on unexpected roles for the SLIT-ROBO signaling pathway in the regulation of podocyte F-actin cytoskeleton and foot process structure in the kidney.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Application ofInternational Application No. PCT/US2013/020280 filed Jan. 4, 2013,which designates the U.S., and which claims benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 61/583,379 filedon 5 Jan. 2012, the contents of which are incorporated herein byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract No.DK078226 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention relates to methods for the treatment ofchronic kidney disease and proteinuria and for the diagnosis of chronickidney disease and monitoring the effects of treatment on theprogression of chronic kidney disease and proteinuria.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 14, 2013, isnamed 701586-072811-PCT_SL.txt and is 50,526 bytes in size.

SUMMARY OF THE INVENTION

Provided herein are novel methods for the treatment of chronic kidneydisease and proteinuria and for the diagnosis of chronic kidney disease,and monitoring the effects of treatment on the progression of chronickidney disease and proteinuria based, in part, on the inventors'discovery of a novel and unexpected role for the SLIT-ROBO signalingpathway in the regulation of podocyte F-actin cytoskeleton and footprocess structure in the kidney.

Accordingly, in some aspects, provided herein are methods for thetreatment of chronic kidney disease in a subject in need thereof, themethods comprising administering to a subject having or at risk for achronic kidney disease a therapeutically effective amount of acomposition comprising a ROBO2 inhibitor.

Also provided herein, in some aspects, are method for the reduction ofproteinuria in a subject in need thereof, comprising administering to asubject having or at risk for proteinuria a therapeutically effectiveamount of a composition comprising a ROBO2 inhibitor.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitor is a blocking antibody or antigen-bindingfragment thereof specific for ROBO2, an antisense molecule specific forROBO2, a short interfering RNA (siRNA) specific for ROBO2, a smallmolecule inhibitor of ROBO2, a ROBO2 inhibitory polypeptide, or a ROBO2structural analog.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitor blocks or reduces binding of ROBO2 to SLIT,to Nck, or to both.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitor is specific for the Ig1 SLIT binding domain,the Ig1 and Ig2 SLIT binding domains, the Nck intracellular bindingdomain, or any combination thereof.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitory polypeptide is a dominant negative ROBO2fusion protein, a polypeptide comprising a ROBO2 extracellular domainwithout the intracellular domain, or a polypeptide comprising a ROBO2intracellular domain without the extracellular domain.

In some embodiments of these methods and all such methods describedherein, the subject having or at risk for a chronic kidney disease hasdiabetic nephropathy or high blood pressure.

In some embodiments of these methods and all such methods describedherein, the method further comprises administering to the subject anadditional therapeutic agent.

In some embodiments of these methods and all such methods describedherein, the additional therapeutic agent is an angiotensin-convertingenzyme (ACE) inhibitor or an angiotensin II receptor blocker (ARB).

Also provided herein, in some aspects, are methods comprising:

-   -   a. assaying a biological test sample from a subject to determine        an expression level of ROBO2 polypeptide or an RNA encoding a        ROBO2 polypeptide;    -   b. determining whether the expression level of ROBO2 polypeptide        or the expression level of the RNA encoding a ROBO2 polypeptide        in the biological test sample is above a reference threshold        level; and    -   c. diagnosing the subject as in need of treatment or therapy for        chronic kidney disease.

In some embodiments of these methods and all such methods describedherein, assaying the expression level of ROBO2 polypeptide is performedusing an antibody or antigen-binding fragment thereof specific for theROBO2 polypeptide.

In some embodiments of these methods and all such methods describedherein, assaying the expression level of the RNA encoding a ROBO2polypeptide is performed using PCR or a hybridization assay.

In some embodiments of these methods and all such methods describedherein, the biological test sample is a kidney biopsy, urine, blood,serum sample, or cells pelleted from a urine sample.

In some embodiments of these methods and all such methods describedherein, the expression level of ROBO2 polypeptide or the expressionlevel of the RNA encoding a ROBO2 polypeptide is at least 20% above thereference threshold level.

In some embodiments of these methods and all such methods describedherein, the expression level of ROBO2 polypeptide or the expressionlevel of the RNA encoding a ROBO2 polypeptide is at least two standarddeviations above the reference threshold level.

Also provided herein, in some aspects, are assays comprising:

-   -   a. contacting a biological test sample isolated from a subject        with a reagent that detects ROBO2 polypeptide or an RNA encoding        a ROBO2 polypeptide; and    -   b. measuring the level of ROBO2 polypeptide or an RNA encoding a        ROBO2 polypeptide,

wherein an increased level of said ROBO2 polypeptide or said RNAencoding a ROBO2 polypeptide, relative to a normal biological sample,identifies a subject having chronic kidney disease and/or progression ofchronic kidney disease or proteinuria.

In some embodiments of these assays and all such assays describedherein, detecting the expression level of ROBO2 polypeptide is performedusing an antibody or antigen-binding fragment thereof specific for theROBO2 polypeptide.

In some embodiments of these assays and all such assays describedherein, detecting the expression level of the RNA encoding a ROBO2polypeptide is performed using PCR or a hybridization assay.

In some embodiments of these assays and all such assays describedherein, the biological test sample is a kidney biopsy, urine, blood,serum sample, or cells pelleted from a urine sample.

In some embodiments of these assays and all such assays describedherein, the expression level of ROBO2 polypeptide or the expressionlevel of the RNA encoding a ROBO2 polypeptide is at least 20% above thereference threshold level.

In some embodiments of these assays and all such assays describedherein, the expression level of ROBO2 polypeptide or the expressionlevel of the RNA encoding a ROBO2 polypeptide is at least two standarddeviations above the reference threshold level.

In some embodiments of these assays and all such assays describedherein, the subject has been diagnosed with diabetes or high bloodpressure.

In some aspects, provided herein are systems for determining if asubject is at risk for chronic kidney disease or proteinuria, or haschronic kidney disease comprising:

-   -   a. a measuring module configured to determine the expression        level of ROBO2 polypeptide or the expression level of the RNA        encoding a ROBO2 polypeptide in a biological sample obtained        from a subject;    -   b. a comparison module configured to receive said expression        level of ROBO2 polypeptide or the expression level of the RNA        encoding a ROBO2 polypeptide determined by the measuring module        and perform at least one analysis to determine whether the        expression level of ROBO2 polypeptide or the expression level of        the RNA encoding a ROBO2 polypeptide is greater than a        pre-determined reference level or ratio, and to provide a        retrieved content; and    -   c. a display module for displaying a content based the data        output from said comparison module, wherein the content        comprises a signal indicative that the expression level or ratio        of ROBO2 polypeptide or RNA is greater than the pre-determined        reference level or ratio, or a signal indicative that the level        or expression ratio of ROBO2 is not greater than the reference        level or pre-determined ratio.

In some embodiments of these systems and all such systems describedherein, the content displayed on the display module further comprises asignal indicative of the subject being recommended to receive aparticular treatment regimen.

In some aspects, provided herein are systems for determining if asubject is at risk for chronic kidney disease or proteinuria, or haschronic kidney disease comprising:

-   -   a. a determination module configured to receive at least one        test sample obtained from a subject and perform at least one        analysis on said at least one test sample to determine the        presence or absence of either of the following conditions:        -   i. an expression ratio of ROBO2 greater than a            pre-determined ratio, or        -   ii. an expression level of ROBO2 greater than a            pre-determined level    -   b. a storage device configured to store data output from said        determination module; and    -   c. a display module for displaying a content based on the data        output from said determination module, wherein the content        comprises a signal indicative that the expression ratio of ROBO2        is greater than the pre-determined ratio or level of ROBO2        greater than a pre-determined level, or a signal indicative that        the expression ratio of ROBO2 is not greater than the        pre-determined ratio or not greater than a pre-determined level.

In some embodiments of these systems and all such systems describedherein, the content displayed on the display module further comprises asignal indicative of the subject being recommended to receive aparticular treatment regimen.

Also provided herein, in some aspects, are methods for treating a humansubject with a risk of chronic kidney disease or proteinuria, comprisingadministering a treatment or therapy to prevent the occurrence ofchronic kidney disease or proteinuria to a human subject who isdetermined to have a level of ROBO2 protein above a reference thresholdlevel.

In some embodiments of these methods and all such methods describedherein, the level of ROBO2 protein is at least 20% above the referencelevel.

In some embodiments of these methods and all such methods describedherein, the level of ROBO2 protein is at least two standard deviationsabove the reference level.

In some embodiments of these methods and all such methods describedherein, the treatment or therapy to prevent the occurrence of chronickidney disease or proteinuria comprises a ROBO2 inhibitor.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitor is a blocking antibody or antigen-bindingfragment thereof specific for ROBO2, an antisense molecule specific forROBO2, a short interfering RNA (siRNA) specific for ROBO2, a smallmolecule inhibitor of ROBO2, a ROBO2 inhibitory polypeptide, or a ROBO2structural analog.

In some embodiments of these methods and all such methods describedherein, ROBO2 inhibitor blocks or reduces binding of ROBO2 to SLIT, toNck, or to both.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitor is specific for the Ig1 SLIT binding domain,the Ig1 and Ig2 SLIT binding domains, the Nck intracellular bindingdomain, or any combination thereof.

In some embodiments of these methods and all such methods describedherein, the ROBO2 inhibitory polypeptide is a dominant negative ROBO2fusion protein, a polypeptide comprising a ROBO2 extracellular domainwithout the intracellular domain, or a polypeptide comprising a ROBO2intracellular domain without the extracellular domain.

Also provided herein, in some aspects, are ROBO2 inhibitors for use intreating a chronic kidney disease, and ROBO2 inhibitor for use intreating proteinuria.

In some embodiments of these uses and all such uses described herein,the ROBO2 inhibitor is a blocking antibody or antigen-binding fragmentthereof specific for ROBO2, an antisense molecule specific for ROBO2, ashort interfering RNA (siRNA) specific for ROBO2, a small moleculeinhibitor of ROBO2, a ROBO2 inhibitory polypeptide, or a ROBO2structural analog.

In some embodiments of these uses and all such uses described herein,the ROBO2 inhibitor blocks or reduces binding of ROBO2 to SLIT, to Nck,or to both.

In some embodiments of these uses and all such uses described herein,the ROBO2 inhibitor is specific for the Ig1 SLIT binding domain, the Ig1and Ig2 SLIT binding domains, the Nck intracellular binding domain, orany combination thereof.

In some embodiments of these uses and all such uses described herein,the ROBO2 inhibitory polypeptide is a dominant negative ROBO2 fusionprotein, a polypeptide comprising a ROBO2 extracellular domain withoutthe intracellular domain, or a polypeptide comprising a ROBO2intracellular domain without the extracellular domain.

In some embodiments of these uses and all such uses described herein,the chronic kidney disease or proteinuria is caused by diabeticnephropathy or high blood pressure.

In some embodiments of any of these aspects and all such aspectsdescribed herein, ROBO2 refers to human ROBO2 having the amino acidsequence of SEQ ID NO: 1 encoded by the mRNA sequence of SEQ ID NO: 2.In some embodiments of any of these aspects and all such aspectsdescribed herein, ROBO2 refers to human ROBO2 having the amino acidsequence of SEQ ID NO: 3 encoded by the mRNA sequence of SEQ ID NO: 4.

DEFINITIONS

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and the include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±10%, ±5%, or ±1%.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

It is understood that the following detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1R demonstrate that Robo2 is expressed and localized to thebasal cell surface of podocytes. All immunostainings in (FIGS. 1A-1Q)are performed at mouse E16.5 days at 600× magnification (see FIGS. 5A-5Mfor immunostainings in adult mouse glomeruli). (FIGS. 1A-1C) Robo2co-localizes with podocyte slit-diaphragm protein nephrin. (FIGS. 1D-1F)Robo2 co-localizes with podocyte slit-diaphragm protein podocin. (FIGS.1G-1I) Robo2 co-localizes with adaptor protein Nck in glomeruli. (FIGS.1J-1L) Robo2 is expressed on the basal podocyte surface adjacent toglomerular basement membrane protein nidogen. (FIGS. 1M-1O) Robo2 doesnot co-localize with glomerular endothelial cell protein marker Pecam1.(FIG. 1P) The enlarged region boxed in (FIG. 1L) shows that Robo2 isexpressed predominantly on the basal cell surface (arrow) of podocytes(p) adjacent to glomerular basement membrane marker nidogen. Robo2 isweakly expressed in the apical and lateral cell surfaces (arrowheads) ofpodocytes. (FIG. 1Q) Robo2 is expressed predominantly on the basal cellsurface (arrows) of podocytes (p) and is weakly expressed on the apicalor lateral cell surfaces (arrowheads). (FIG. 1R) Immunogold electronmicroscopy shows localization of gold partials (arrows) conjugated toRobo2 antibody in the foot process (fp) of a podocyte from a 3-week oldmouse. GBM, glomerular basement membrane. Magnification: 50,000×. Seealso FIGS. 5A-5M.

FIGS. 2A-2J demonstrate that Robo2 interacts with the adapter proteinNck and forms a complex with nephrin. (FIG. 2A) Yeast two-hybrid assaysshow a positive interaction between Robo2 intracellular domain(Robo2-ICD) and Nck1. LacZ reporter (X-gal): +++, yeast turned dark; ++,light; −, white in 24 hours. Leucine reporter (−Leu): +, yeast grew; −,yeast did not grow. CC, cytoplasmic conserved region. Numbers indicateresidue positions in the full-length protein. (FIG. 2B) Yeast two-hybridassays show the first two SH3 domains of Nck1 are required for itsinteraction with Robo2-ICD. (FIG. 2C, FIG. 2C discloses SEQ ID NO: 5)Yeast two-hybrid assays show potential binding domains that mediateRobo2 and Nck1 interaction. The sequence is the potential binding regionin Robo2 for Nck1. Proline-rich regions are highlighted. (FIG. 2D)Co-precipitation of Robo2 and Nck. Cell lysates in lane 5 are collectedfrom His-myc-Robo2 transfected cells (used in lanes 1 and 2); Celllysates in lane 6 are collected from His-myc-Robo2-ANBD transfectedcells (used in lanes 3 and 4). (FIG. 2E) Co-precipitation of Robo2, Nck,and nephrin. (FIG. 2F) A similar co-precipitation as (FIG. 2E) exceptthat His-myc-nephrin is pulled-down instead of His-myc-Robo2. (FIG. 2G)Co-immunoprecipitation of kidney endogenous Robo2, Nck, and nephrin.(FIG. 2H) A similar assay as (FIG. 2G) except that precipitates areprepared using mouse anti-nephrin antibody. (I) Slit2 enhancesRobo2-Nck-nephrin complex formation. His-myc-Robo2, nephrin, and Fyn areexpressed in HEK cells that are stimulated with Slit2 conditioned medium(lanes 1, 3) or control conditioned medium (lane 2, 4). (FIG. 2J)Intensity quantification of (FIG. 2I). Data are represented as mean±SEM;n=7, *p<0.05, **p<0.01 compared with the control, paired student'st-test. See also FIGS. 6A-6I.

FIGS. 3A-3G demonstrate that Slit2-Robo2 signaling inhibitsnephrin-mediated actin polymerization. (FIG. 3A) CD16/7-NCD isco-expressed with Robo2 in HEK cells, which are treated with anti-CD16antibody and rhodamine-conjugated anti-IgG antibody in the presence ofSlit2 conditioned medium (Slit) or control conditioned medium (CTL).Cells are then fixed and stained with FITC-conjugated phalloidin toreveal F-actin. Scale bar, 5 μm. NCD: nephrin cytoplasmic domain. (FIG.3B) A similar assay as (FIG. 3A) except that CD16/7-NCD is replaced byCD16/7-HA and is used as a control assay. (FIG. 3C) The percentage ofcells with F-actin tails over total cells with CD16/7 clusters in eachgroup is quantified. Data are represented as mean±SEM, *p<0.01, n=5.(FIG. 3D) CD16/7-NCD in (FIG. 3A) is immunoprecipitated by anti-CD16antibody after Slit2 conditioned medium stimulation (lanes 1 and 3) orcontrol conditioned medium (lanes 2 and 4). Note reduced F-actin inlane 1. CD16/7-HA is used as a negative control. (FIG. 3E) Intensityquantification of (FIG. 3D). Data are represented as mean±SEM; n=4,*p<0.05 compared with the control, paired student's t-test. (FIG. 3F)Immunoprecipitation of nephrin from Robo2 knockout homozygous(Robo2−/−), heterozygous (Robo2+/−), and wild-type (Robo2+/+) mousekidneys using the anti-nephrin antibody. Note increased F-actin in lane3. (FIG. 3G) Intensity quantification of (FIG. 3F). Data are representedas mean±SEM; n=4, *p<0.05 compared with the wild-type and heterozygous,ANOVA analysis. See also FIGS. 7A-7C.

FIGS. 4A-4W demonstrate podocyte structural phenotypes in the Robo2homozygous null, Robo2 podocyte specific knockout, and Robo2 and Nphs1double knockout mice. (FIGS. 4A and 4B) Representative images of newbornkidneys show podocyte bodies (arrowheads) and Bowman's capsule (arrows)in wild-type (FIG. 4A) and Robo2 homozygous null mice (FIG. 4B). (FIGS.4C and 4D) High magnification images of (FIGS. 4A and 4 B) show podocytefoot processes (arrows) in the newborn kidney. Scale bar, 1 μm. (FIGS.4E and 4F) Representative images of 3-week kidneys at low magnificationshow podocyte cell body (arrowheads) in a Robo2 homozygous null mouse(FIG. 4F) compared to an age-matched control (FIG. 4E). (FIGS. 4G and4H) Higher magnification images of (FIGS. 4E and 4F) show disorganizedshorter meandering foot processes (arrow) in a 3-week Robo2 homozygousnull mouse (FIG. 4H) compared to well-organized zip-like foot processesin the age-matched control (FIG. 4G). Scale bars: 2 μm. (FIGS. 4I and4J) Representative transmission electron microscopy images(magnification at 5000×) depict the focal segmental podocyte footprocess effacement (arrow in FIG. 4J) in a one month old Robo2podocyte-specific knockout mouse and the normal phenotype in the control(FIG. 4I). Abbreviations: gc: glomerular capillary; us: urinary space.(FIGS. 4K and 4L) Higher magnification transmission electron microscopyimages (40000×) show broader podocyte foot processes (arrow in FIG. 4L)in a two months old Robo2 podocyte-specific mutant mouse compared withthe control (FIG. 4K). Abbreviations: fp, podocyte foot process; GBM,glomerular basement membrane. (FIG. 4M) Quantification of podocyte footprocess width in one month old Robo2^(del5/flox); Tg^(Nphs2-Cre+)podocyte specific knockout mice (Robo2 KO) and the wild-type littermatecontrols (WT). Data are represented as mean±SEM, n=333, *p<0.01. (FIG.4N) ELISA assay of spot urine shows an elevated albumin/creatinine ratioin Robo2^(del5/flox); Nphs2-Cre+ (KO) adult mice compared with controlwild-type (WT). Data are represented as mean±SEM, n=20, *p<0.01. (FIG.4O) Western blot analysis shows the presence of albumin in urine; 1 μlurine was loaded on each well, 0.2 μg albumin was used as a positivecontrol. WT, three wild-type littermates; Robo2 KO, three individualRobo2del5/flox; Nphs2-Cre+ mice. (FIGS. 4P and 4Q) Representativescanning electron microscope images show disrupted interdigitatingpodocyte foot processes that resemble disorganized cellular protrusions(arrows) in the Nphs1−/− single homozygous newborn mouse kidney. Scalebars: 1 μm. (FIGS. 4R and 4S) Glomeruli from Nphs1−/−Robo2−/− doublehomozygous newborn mice exhibit restored interdigitating foot processes(arrows), indicating alleviation of nephrin null phenotype by knockingout Robo2. (FIGS. 4T and 4U), Glomeruli from Robo2−/− single homozygousnewborn mice display irregular and broader foot processes but extensiveinterdigitating pattern formation (arrows). (FIGS. 4V and 4W), Glomerulifrom newborn wild type mice with normal regular interdigitating patternof the foot process (arrows). See also FIGS. 8A-8Z and Tables 1-4.

FIGS. 5A-5M demonstrate that Robo2 is expressed in the developing andadult glomeruli. (FIGS. 5A and 5B) In situ hybridization analysis showsthat Robo2 transcripts are expressed in developing glomeruli (arrows) atE16.5. Magnification: 60× (FIG. 5A) and 200× (FIG. 5B). (FIGS. 5C-5F)Immunohistochemistry (IHC) studies reveal that Robo2 is expressed duringdeveloping glomeruli from E14.5 to E17.5. Magnification: 600×. (FIG. 5G)IHC shows that Robo2 is specifically expressed in adult mouse glomeruliat 5 weeks of age (FIG. 5G). DAPI marks cell nuclei in the kidney.Magnification: 400×. (FIG. 5H) IHC co-localization stainings of 5 wkidney show Robo2 is co-expressed in the glomerulus with podocyte markerWt1. Magnification: 600×. (FIGS. 5I-5K) Robo2 and WT1 are co-expressedin the mouse glomerulus at E16.5. Magnification: 600×. (FIGS. 5L and 5M)IHC co-localization stainings of 5 w kidney show Robo2 is co-expressedin the glomerulus with mesangial cell marker Pdgfrb (FIG. 5L), andendothelial cell marker Pecam1 (FIG. 5M). Magnification: 600×.

FIGS. 6A-6I demonstrate that Robo2 interacts with Nck and forms acomplex with nephrin, which is enhanced by Slit2 stimulation. (FIG. 6A)Co-IP of Robo2 and nephrin with endogenous Nck. Robo2, nephrin, and Fynare expressed in HEK cells and stimulated by Slit2. The endogenous Nckis immunoprecipitated by an anti-Nck antibody. The mouse IgG is used asa control. The complex formation with nephrin is enhanced by Slit2 andFyn expression. (FIGS. 6B and 6C) Slit2 is expressed in the newbornmouse glomeruli by Immunoperoxidase staining (FIG. 6B) and isco-expressed in the glomerulus with the podocyte marker Synaptopodin(FIG. 6C). Magnification: 600×. (FIGS. 6D and 6E) CD16/7-NCD isco-expressed with Robo2 in HEK cells in the presence of Slit2, treatedwith anti-CD16 antibody and rhodamine-conjugated anti-IgG antibody, thenfixed and stained with anti-Robo2 antibody. CD16/7-NCD clustersco-localize with Robo2 (FIG. 6D) but no colocalization is observed incontrol CD16/7-HA clusters (FIG. 6E). Scale bar: 5μπι. NCD: nephrincytoplasmic domain. (FIGS. 6F and 6G) Deletion of Nck binding domain(NBD) in Robo2 impairs its co-localization with CD16/7-NCD in thepresence of Slit2. CD 16/7-NCD clusters co-localize with Robo2 (FIG. 6F)but no colocalization is observed in Robo2-ΔNBD clusters (FIG. 6G).Scale bar: 5μπι. (FIGS. 6H and 6I) Slit2 stimulation enhances CD16/7-NCDand Robo2 co-localization in HEK cells. CD 16/7-NCD clusters co-localizewith Robo2 in the presence of Slit2 (FIG. 6H) but not with controlconditioned medium (FIG. 6I). Scale bar: 5μπι.

FIGS. 7A-7C demonstrate deletion of Nck binding domain in Robo2compromises Slit2-Robo2 inhibition on nephrin-induced actinpolymerization. (FIG. 7A) CD16/7-NCD and Robo2 were co-expressed in HEKcells, clustered with anti-CD16 antibody and rhodamine-conjugatedanti-IgG antibody in the presence of Slit2 conditioned medium (Slit2) orcontrol conditioned medium (CTL). Cells were then fixed and stained withFITC-conjugated phalloidin to reveal F-actin fibers. Clusters ofCD16/7-NCD and F-actin fibers were examined using confocal microscopy.Scale bar, 5 μm. NCD, nephrin cytoplasmic domain. (FIG. 7B) CD16/7-NCDand Robo2-ΔNBD were co-expressed in HEK cells. Scale bar, 5 μm. NBD, Nckbinding domain. (FIG. 7C) The percentage of cells with F-actin tailsover total cells with CD16/7-NCD clusters in each group was quantified.Data are represented as mean±SEM, *p=1.436×10⁻⁵, **p=6.32×10⁻⁵, n=5,ANOVA.

FIGS. 8A-8Z demonstrate glomerular phenotype in the Robo2 homozygousnull, Robo2 podocyte specific knockout, Robo2 and Nphs1 double knockoutmice, and a proposed model of Robo2-Nephrin signaling. (FIGS. 8A-8F)Transmission electron microscopy analysis of glomerular ultrastructurein newborn (NB) Robo2^(del5/del5) mutant mice kidney. (FIGS. 8A, 8C, 8E)Glomerular ultrastructure from a newborn heterozygous Robo2 controlmouse at low (FIG. 8A, 2200×), medium (FIG. 8C, 15500×) and high (FIG.8E, 52000×) magnifications. (FIGS. 8B, 8D, 8F) Glomerular ultrastructurefrom a newborn homozygous Robo2 (−/−) (i.e., Robo2^(del5/del5)) mutantmouse at low (FIG. 8B), medium (FIG. 8D) and high (FIG. 8F)magnifications. Arrows indicate focal foot process effacement.Abbreviations: gc: glomerular capillary; us: urinary space; GBM:glomerular basement membrane. (FIGS. 8G-8N) Abnormal podocyte footprocess patterns in Robo2 podocyte-specific knockout mice. (FIGS. 8G-8J)Representative scanning electron microscopy images of glomeruli fromone-month old Robo2^(del5/flox); Nphs2-Cre⁺ podocyte-specific knockoutmice and aged matched Robo2^(flox/+) control mice. Mild irregularitiesof the interdigitating podocyte foot processes were found in a one monthold Robo2 podocyte-specific knockout mouse (FIGS. 8K and 8N). At sevenmonths old, Robo2 podocyte-specific knockout mice developed markedlyirregular foot processes (FIGS. 8L and 8N). Scale bars: 10 μm (FIGS. 8G,8H, 8K, 8L at 2000× magnification) and 2 μm (FIGS. 8I, 8J, 8M, 8N at13000× magnification). (FIGS. 8O-8T) Glomerular morphology in Robo2podocyte-specific knockout mice. (FIGS. 8O-8R) Periodic acid-Schiff(PAS) staining showed mesangial matrix expansion in the glomeruli from2-month and 6-month old Robo2 podocyte-specific knockout mice (FIGS. 8P,8R) compared to age-matched controls (FIGS. 8O, 8Q). (FIG. 8S)Quantitative analysis of glomeruli shows mesangial matrix expansion in12-month old Robo2 podocyte-specific knockout mice (MU) compared to agematched wild-type (WT) controls. Data are represented as mean±SEM, n=5,*p<0.01. (T) Robo2 podocyte specific knockout does not affect podocytenumbers. Podocyte cells were identified using WT-1 staining. The numberof podocytes per glomerular cross section was counted in four one-yearold Robo2^(del5/flox); Tg^(Nphs2-Cre+) podocyte specific knockout mice(MU) compared to four age-matched wild-type mice (WT). Data arerepresented as mean±SEM, p=0.645, t-test; mutant: n=165 glomeruli;control: n=166 glomeruli. (FIGS. 8U-8Y) Glomerular phenotype in Robo2and Nphs1 double knockout mice. (FIG. 8U) H&E staining shows glomeruliwith characteristic dilatations of the Bowman's space (asterisks) in aNphs1^(−/−) single homozygous newborn mouse, 400×. (FIG. 8V) Glomerulifrom a Robo2^(−/−) single homozygous newborn mouse show absence ofBowman's space dilatations; 400×. (FIG. 8W) Normal looking glomeruliwithout significant Bowman's space dilatations (arrows) are shown in aRobo2^(−/−); Nphs1^(−/−) double homozygous newborn mouse indicatingalleviation of Nphs1^(−/−) glomerular phenotype; 400×. (FIG. 8X) H&Estaining of normal kidney and glomeruli from an age-matched wild-typenewborn mouse control; 400×. (FIG. 8Y) Quantification of glomeruli withdilated Bowman's space in newborn mice show significant reduction ofglomeruli with the characteristic dilatation phenotype of the Bowman'sspace in Robo2^(−/−); Nphs1^(−/−) double homozygous compared toNphs1^(−/−) single homozygous (Robo2^(+/−); Nphs1^(−/−)). Data arerepresented as mean±SEM, *p<0.01. (FIG. 8Z) A model of inhibitoryeffects of Slit2-Robo2 signaling on nephrin to influence podocyte footprocess structure: Under physiological conditions (e.g., during footprocess development), nephrin intracellular phosphorylated tyrosinedomains (YDxV-p) recruit Nck through its interaction with the SH2domain. Nck in turn recruits cytoskeleton regulators through its SH3domains to promote actin polymerization. Slit2 binds Robo2 to increaseRobo2 intracellular domain interaction with SH3 domains of Nck, whichwould prevent binding of Nck to cytoskeletal regulators and result in aninhibition of nephrin-induced actin polymerization. Balanced actinpolymerization is maintained during podocte development for a normalfoot process structure. In the absence of Slit2-Robo2 signaling (e.g.,when Robo2 is knocked out), the inhibitory effects of Robo2 on nephrininduced polymerization is lost. The SH3 domains of Nck are able tointeract with downstream cytoskeletal regulators to increase actinpolymerization, which may explain the altered podocyte foot processstructure in Robo2 mutant mice. Abbreviations: Ig: Immunoglobulindomain; FN3: Fibronectin type 3 domain; SH2: Src homolog 2 domain; SH3:Src homolog 3 domain; CC0, CC1, CC2, CC3: Cytoplasmic Conserved region0, 1, 2, 3.

DETAILED DESCRIPTION

Robo2 has been previously shown to be the cell surface receptor for therepulsive guidance cue Slit and to be involved in axon guidance andneuronal migration in the nervous system. Nephrin is a podocyteslit-diaphragm protein that functions in the kidney glomerularfiltration barrier. We demonstrate herein that Robo2 is expressed at thebasal surface of podocytes, such as mouse podocytes, and co-localizeswith nephrin. Biochemical studies indicate that Robo2 forms a complexwith nephrin in the kidney through adaptor protein Nck. In contrast tothe role of nephrin that promotes actin polymerization, we show hereinthat Slit2-Robo2 signaling inhibits nephrin-induced actinpolymerization. For example, the amount of F-actin associated withnephrin is increased in Robo2 knockout mice that develop an alteredpodocyte foot process structure and microalbuminuria. Geneticinteraction studies further reveal that loss of Robo2 alleviates theabnormal podocyte phenotype in nephrin null mice. The results providedherein show that Robo2 signaling acts as a negative regulator on nephrinto influence podocyte foot process architecture.

In addition, it has been shown that a patient having vesicoureteralreflux (VUR) has a chromosome translocation that disrupts the ROBO2 geneand produces dominant negative ROBO2 fusion proteins that abrogate theSLIT2-ROBO2 signaling pathway. Normally, VUR is a disease characterizedby the retrograde flow of urine from the bladder into the ureters andkidney and VUR patients can present with reflux nephropathy, a conditionthat manifests with severe proteinuria. It has been shown that dominantnegative ROBO2 fusion proteins produced by a VUR patient blocks theSLIT2-ROBO2 signaling pathway and protects the patient from refluxnephropathy and proteinuria, thus confirming and further supporting theinventors results in animal models of the therapeutic value of targetingthe SLIT2-ROBO2 signaling pathway for the treatment of chronic kidneydisease.

In the normal kidney, the trilaminar glomerular capillary wall, composedof fenestrated endothelial cells, basement membrane and podocytes,restricts the permeability to plasma proteins. Podocytes are specializedepithelial cells that extend primary and secondary processes to coverthe outer surface of the glomerular basement membrane. The actin-richinterdigitating secondary processes, or foot processes, from neighboringpodocytes create filtration slits bridged by a semi-porousslit-diaphragm that forms the final barrier to protein permeation.Whereas genetic mutations of podocyte slit-diaphragm proteins such asnephrin and others are associated with hereditary forms of proteinurickidney disease (Tryggvason et al., 2006), it has become evident that theproteins that make up and associate with the slit-diaphragm are morethan a simple structural barrier. These proteins form a balancedsignaling network that can influence podocyte foot process structure andfunction through interaction with the F-actin cytoskeleton (Faul et al.,2007; Jones et al., 2006; Verma et al., 2006).

Roundabout (Robo) family proteins, Robo1, Robo2, Robo3 and Robo4 arecell surface receptors for the secreted ligand Slit (Dickson andGilestro, 2006). Slit1, Slit2, and Slit3 were originally found asrepulsive guidance cues for axon pathfinding and migrating neuronsduring nervous system development (Guan and Rao, 2003). Thetransmembrane protein Robo contains five Ig motifs and three fibronectintype III (FNIII) repeats in its extracellular domain (Dickson andGilestro, 2006). While both immunoglobulin (Ig) motifs 1 and 2 interactwith Slit, the first Ig1 motif of Robo is the primary binding site forSlit (Dickson and Gilestro, 2006). The intracellular domain of Robo hasfour cytoplasmic conserved (CC) sequences named CC0, CC1, CC2, and CC3(Bashaw et al., 2000; Kidd et al., 1998; Morlot et al., 2007; Zallen etal., 1998). CC0 and CC1 contain tyrosine, while CC2 and CC3 areproline-rich stretches. The repulsive activity of Slit-Robo signalinginhibits actin polymerization (Guan and Rao, 2003) or induces F-actindepolymerization (Piper et al., 2006).

Slit-Robo signaling also plays crucial roles during early kidneyinduction and ureteric bud outgrowth. Mouse mutants that lack Slit2 orRobo2 develop supernumerary ureteric buds, which lead to abroad-spectrum of urinary tract phenotype including duplex kidneys,abnormal ureterovesical junctions and hydronephrosis (Grieshammer etal., 2004; Lu et al. 2007). Disruption of ROBO2 in humans causescongenital anomalies of the kidneys and urinary tracts (CAKUT), andpoint mutations of ROBO2 have been identified in patients withvesicoureteral reflux (VUR) (Lu et al., 2007). Our recent studydemonstrates that Robo2 is crucial for the formation of a normalureteral orifice and for the maintenance of an effective anti-refluxmechanism (Wang et al., 2011).

Herein we demonstrate that Robo2 is a novel podocyte protein expressedat the basal surface of glomerular podocytes in the kidney and isco-localized with nephrin and podocin. Robo2 interacts directly withadaptor protein Nck SH3 domains and forms a complex with nephrin.Whereas Robo2 knockout mice develop altered podocyte foot processes, theloss of Robo2 alleviates the foot process structural abnormalities thatare seen in nephrin null mice. These results described herein indicatethat Robo2 signaling acts as a negative regulator on nephrin signalingto influence podocyte foot process architecture. In addition, asdemonstrated herein, it has been discovered that the dominant negativeROBO2 fusion proteins produced by a patient blocks the SLIT2-ROBO2signaling pathway and protects the patient from reflux nephropathy andproteinuria, thus confirming and further supporting the resultsdescribed herein in animal models of the therapeutic value of targetingthe SLIT2-ROBO2 signaling pathway for the treatment of chronic kidneydisease.

Accordingly, in some aspects, provided herein are methods for thetreatment of chronic kidney disease in a subject in need thereof, suchmethod comprising administering to a subject having or at risk for achronic kidney disease a therapeutically effective amount of acomposition comprising a SLIT2-ROBO2 signaling pathway inhibitor.

Also provided herein, in some aspects, are methods for the reduction ofproteinuria in a subject in need thereof, comprising administering to asubject having or at risk for proteinuria a therapeutically effectiveamount of a composition comprising a SLIT2-ROBO2 signaling pathwayinhibitor.

In other aspects, provided herein are methods for preventing kidneydiseases or promoting prophylaxis of kidney diseases in a subject inneed thereof, comprising administering to a subject a therapeuticallyeffective amount of a composition comprising a SLIT2-ROBO2 signalingpathway inhibitor so as to prevent or promote prophylaxis of kidneydisease in the subject.

Also provided herein, in some aspects, are methods for mitigating theeffects of kidney disease, reducing the severity of kidney disease,reducing the likelihood of developing kidney disease and/or slowing theprogression of kidney disease in a subject in need thereof.

As used herein, “ROBO2” refers to the polypeptide having the amino acidsequence of:MARRHERVTRRMWTWAPGLLMMTVVFWGHQGNGQGQGSRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVTDVLTDRPPPIILQGPANQTLAVDGTALLKCKATGDPLPVISWLKEGFTFPGRDPRATIQEQGTLQIKNLRISDTGTYTCVATSSSGETSWSAVLDVTESGATISKNYDLSDLPGPPSKPQVTDVTKNSVTLSWQPGTPGTLPASAYIIEAFSQSVSNSWQTVANHVKTTLYTVRGLRPNTIYLFMVRAINPQGLSDPSPMSDPVRTQDISPPAQGVDHRQVQKELGDVLVRLHNPVVLTPTTVQVTWTVDRQPQFIQGYRVMYRQTSGLQATSSWQNLDAKVPTERSAVLVNLKKGVTYEIKVRPYFNEFQGMDSESKTVRTTEEAPSAPPQSVTVLTVGSYNSTSISVSWDPPPPDHQNGIIQEYKIWCLGNETRFHINKTVDAAIRSVIIGGLFPGIQYRVEVAASTSAGVGVKSEPQPIIIGRRNEVVITENNNSITEQITDVVKQPAFIAGIGGACWVILMGFSIWLYWRRKKRKGLSNYAVTFQRGDGGLMSNGSRPGLLNAGDPSYPWLADSWPATSLPVNNSNSGPNEIGNFGRGDVLPPVPGQGDKTATMLSDGAIYSSIDFTTKTSYNSSSQITQATPYATTQILHSNSIHELAVDLPDPQWKSSIQQKTDLMGFGYSLPDQNKGNNGGKGGKKKKNKNSSKPQKNNGSTWANVPLPPPPVQPLPGTELEHYAVEQQENGYDSDSWCPPLPVQTYLHQGLEDELEEDDDRVPTPPVRGVASSPAISFGQQSTATLTPSPREEMQPMLQAHLDELTRAYQFDIAKQTWHIQSNNQPPQPPVPPLGYVSGALISDLETDVADDDADDEEEALEIPRPLRALDQTPGSSMDNLDSSVTGKAFTSSQRPRPTSPFSTDSNTSAALSQSQRPRPTKKHKGGRMDQQPALPHRREGMTDEEALVPYSKPSFPSPGGHSSSGTASSKGSTGPRKTEVLRAGHQRNASDLLDIGYMGSNSQGQFTGEL (Homo sapiens roundabout homolog 2 isoform ROBO2a;SEQ ID NO: 1), as described by, e.g., NP_001122401.1 and encoded byNM_001128929.2 (SEQ ID NO: 2); orMSLLMFTQLLLCGFLYVRVDGSRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVTDVLTDRPPPIILQGPANQTLAVDGTALLKCKATGDPLPVISWLKEGFTFPGRDPRATIQEQGTLQIKNLRISDTGTYTCVATSSSGETSWSAVLDVTESGATISKNYDLSDLPGPPSKPQVTDVTKNSVTLSWQPGTPGTLPASAYIIEAFSQSVSNSWQTVANHVKTTLYTVRGLRPNTIYLFMVRAINPQGLSDPSPMSDPVRTQDISPPAQGVDHRQVQKELGDVLVRLHNPVVLTPTTVQVTWTVDRQPQFIQGYRVMYRQTSGLQATSSWQNLDAKVPTERSAVLVNLKKGVTYEIKVRPYFNEFQGMDSESKTVRTTEEAPSAPPQSVTVLTVGSYNSTSISVSWDPPPPDHQNGIIQEYKIWCLGNETRFHINKTVDAAIRSVIIGGLFPGIQYRVEVAASTSAGVGVKSEPQPIIIGRRNEVVITENNNSITEQITDVVKQPAFIAGIGGACWVILMGFSIWLYWRRKKRKGLSNYAVTFQRGDGGLMSNGSRPGLLNAGDPSYPWLADSWPATSLPVNNSNSGPNEIGNFGRGDVLPPVPGQGDKTATMLSDGAIYSSIDFTTKTSYNSSSQITQATPYATTQILHSNSIHELAVDLPDPQWKSSIQQKTDLMGFGYSLPDQNKGNNGGKGGKKKKNKNSSKPQKNNGSTWANVPLPPPPVQPLPGTELEHYAVEQQENGYDSDSWCPPLPVQTYLHQGLEDELEEDDDRVPTPPVRGVASSPAISFGQQSTATLTPSPREEMQPMLQAHLDELTRAYQFDIAKQTWHIQSNNQPPQPPVPPLGYVSGALISDLETDVADDDADDEEEALEIPRPLRALDQTPGSSMDNLDSSVTGKAFTSSQRPRPTSPFSTDSNTSAALSQSQRPRPTKKHKGGRMDQQPALPHRREGMTDEEALVPYSKPSFPSPGGHSSSGTASSKGSTGPRKTEVLRAGHQRNASDLLDIGYMGSNSQGQFTG EL (Homosapiens roundabout homolog 2 isoform ROBO2b; SEQ ID NO: 3), as describedby, e.g., NP_002933.1 and encoded by NM_002942.4 (SEQ ID NO: 4),together with any naturally occurring allelic, splice variants, andprocessed forms thereof. Typically, ROBO2 refers to human ROBO2. TheROBO2 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse,rat, chicken, zebrafish, fruit fly, mosquito, and C. elegans. Specificresidues of ROBO2 can be referred to as, for example, “ROBO2(30).”

Specific domains of ROBO2 can be referred to by such nomenclature aswell. The N-terminal or “extracellular domain of ROBO2”, comprising thefive immunoglobulin motifs and three fibronectin type III (FNIII)repeats can be referred to as ROBO2(46-848) of SEQ ID NO: 1 orROBO2(30-832) of SEQ ID NO: 3, for example. The immunoglobulin (Ig)motifs 1 and 2 that interact with Slit2, or the “Ig SLIT binding domain”can be referred to as ROBO2(46-145) and ROBO2(151-237) respectively ofSEQ ID NO: 1, and ROBO2(30-129) and ROBO2(135-221) respectively of SEQID NO: 3. Similarly, the “intracellular domain” comprising the “Nckintracellular binding domain,” which comprises the four intracellularproline rich motifs, described herein, can be referred to asROBO2(881-1378) of SEQ ID NO: 3.

As used herein, the terms “ROBO2 inhibitor,” “ROBO2 antagonist,” “ROBO2inhibitor agent,” and “ROBO2 antagonist agent” refer to a molecule oragent that significantly blocks, inhibits, reduces, or interferes withROBO2 (mammalian, such as human, ROBO2) biological activity in vitro, insitu, and/or in vivo, including activity of downstream pathways mediatedby ROBO2 signaling, such as, for example, ROBO2 interaction with theadaptor protein Nck and/or complex formation with nephrin, SLIT2-ROBO-2mediated inhibition of nephrin-mediated actin polymerization, and/orelicitation of a cellular response to ROBO2. The term “agent” as usedherein in reference to a ROBO2 inhibitor means any compound or substancesuch as, but not limited to, a small molecule, nucleic acid,polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical,entity, or moiety, including, without limitation, synthetic andnaturally-occurring proteinaceous and non-proteinaceous entities. Insome embodiments of the aspects described herein, an agent is a nucleicacid, a nucleic acid analogue, a protein, an antibody, a peptide, anaptamer, an oligomer of nucleic acids, an amino acid, or a carbohydrate,and includes, without limitation, proteins, oligonucleotides, ribozymes,DNAzymes, glycoproteins, antisense RNAs, siRNAs, lipoproteins, aptamers,and modifications and combinations thereof etc. Compounds for use in thetherapeutic compositions and methods described herein can be known tohave a desired activity and/or property, or can be selected from alibrary of diverse compounds, using screening methods known to one ofordinary skill in the art.

Exemplary ROBO2 inhibitors contemplated for use in the various aspectsand embodiments described herein include, but are not limited to,anti-ROBO2 antibodies or antigen-binding fragments thereof thatspecifically bind to ROBO2; anti-sense molecules directed to a nucleicacid encoding ROBO2 (e.g., ROBO2a or ROBO2b or both); short interferingRNA (“siRNA”) molecules directed to a nucleic acid encoding ROBO2 (e.g.,ROBO2a or ROBO2b or both); RNA or DNA aptamers that bind to ROBO2, andinhibit/reduce/block ROBO2 mediated signaling; ROBO2 structural analogs;and soluble ROBO2 proteins, inhibitory polypeptides, e.g., dominantnegative polypeptides, or fusion polypeptides thereof. In someembodiments of these aspects and all such aspects described herein, aROBO2 inhibitor (e.g., an antibody or antigen-binding fragment thereof)binds (physically interacts with) ROBO2, targets downstream ROBO2signaling, and/or inhibits (reduces) ROBO2 synthesis, production orrelease. In some embodiments of these aspects and all such aspectsdescribed herein, a ROBO2 inhibitor binds and prevents its binding aSLIT protein ligand, such as SLIT2. In some embodiments of these aspectsand all such aspects described herein, a ROBO2 inhibitor specificallyreduces or eliminates expression (i.e., transcription or translation) ofone or more ROBO2 isoforms.

As used herein, a ROBO2 inhibitor or antagonist has the ability toreduce the activity and/or expression of ROBO2 in a cell (e.g.,podocytes) by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or more, relative to the activity or expressionlevel in the absence of the ROBO2 inhibitor.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, the ROBO2 inhibitor inhibits ROBO2 mediated signaltransduction. In some embodiments of the compositions and methodsdescribed herein, the ROBO2 inhibitor targets ROBO2 interaction with theadaptor protein Nck and/or complex formation with nephrin, SLIT2-ROBO-2mediated inhibition of nephrin-mediated actin polymerization, and/orelicitation of a cellular response to ROBO2.

In some embodiments of the compositions and methods described herein,the binding sites of the ROBO2 inhibitors, such as an antibody orantigen-binding fragment thereof, are directed against a ROBO2 ligandinteraction site, such as a SLIT2 ligand interaction site. In someembodiments of the compositions and methods described herein, thebinding sites of the ROBO2 inhibitor, such as an antibody orantigen-binding fragment thereof, are directed against a ROBO2 adaptorinteraction site such as an Nck interaction site or the NCKintracellular binding domain comprising the four intracellular prolinerich motifs of ROBO2. In some embodiments of the compositions andmethods described herein, the binding sites of the ROBO2 inhibitors aredirected against a site on a target in the proximity of the ligandinteraction site, in order to provide steric hindrance for theinteraction of the receptor (e.g., ROBO2) with its ligand (e.g., SLIT2).By binding to a ROBO2 ligand interaction site, a ROBO2 inhibitordescribed herein can reduce or inhibit the activity or expression ofROBO2, and downstream ROBO2 signaling consequences (e.g., ROBO2interaction with the adaptor protein Nck and/or complex formation withnephrin, SLIT2-ROBO-2 mediated inhibition of nephrin-mediated actinpolymerization, and/or elicitation of a cellular response to ROBO2). Forexample, in some embodiments of the compositions and methods describedherein, the binding sites of the ROBO2 inhibitors block or target atleast the Ig1, and preferably both the Ig1 and Ig2 sites, on ROBO2,i.e., ROBO2(46-145) and ROBO2(151-237) respectively of SEQ ID NO: 1, andROBO2(30-129) and ROBO2(135-221) respectively of SEQ ID NO: 3, forexample. In some embodiments of the compositions and methods describedherein, the binding sites of the ROBO2 inhibitors block or target theROBO2 intracellular domain comprising the Nck intracellular bindingdomain, i.e., ROBO2(881-1378) of SEQ ID NO: 3. In some embodiments ofthe compositions and methods described herein, the binding sites of theROBO2 inhibitors block or target the ROBO2 Nck intracellular bindingdomain comprising the four intracellular proline rich motifs of ROBO2.This can be accomplished by a variety of means well known in the art,such as antibodies and antigen-binding fragments thereof, inhibitorRNAs, etc., and as described herein.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, the ROBO2 inhibitor is an antibody or antigen-bindingfragment thereof that selectively binds or physically interacts withROBO2. In some embodiments of the compositions and methods describedherein, the ROBO2 inhibitor is an antibody or antigen-binding fragmentthereof that binds to ROBO2 and inhibits and/or blocks and/or preventsinteraction with Nck and/or complex formation with nephrin. In someembodiments of the compositions and methods described herein, theantibody or antigen-binding fragment thereof binds to the Ig SLITbinding domain of ROBO2. In some embodiments of the compositions andmethods described herein, the antibody or antigen-binding fragmentthereof binds to the Ig1SLIT binding domain of ROBO2 or both the Ig1 andIg2 SLIT binding domains of ROBO2, i.e., ROBO2(46-145) andROBO2(151-237) respectively of SEQ ID NO: 1, and ROBO2(30-129) andROBO2(135-221) respectively of SEQ ID NO: 3. In some embodiments of thecompositions and methods described herein, the antibody orantigen-binding fragment thereof binds to or blocks the ROBO2intracellular domain, i.e., ROBO2(881-1378) of SEQ ID NO: 3. In someembodiments of the compositions and methods described herein, theantibody or antigen-binding fragment thereof binds to or blocks the Nckintracellular binding domain comprising the four intracellular prolinerich motifs of ROBO2.

Antibodies specific for or that selectively bind ROBO2, suitable for usein the compositions and for practicing the methods described herein arepreferably monoclonal, and can include, but are not limited to, human,humanized or chimeric antibodies, comprising single chain antibodies,Fab fragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, and/or binding fragments of any of the above. Antibodies alsorefer to immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain antigen or targetbinding sites or “antigen-binding fragments.” The immunoglobulinmolecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass of immunoglobulin molecule, as is understood by one of skill inthe art.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, a ROBO2 inhibitor as described herein is a monoclonalanti-ROBO2 antibody or antigen-binding fragment.

In some embodiments of the compositions and methods described herein, aROBO2 inhibitor as described herein is a ROBO2 antibody fragment orantigen-binding fragment. The terms “antibody fragment,” “antigenbinding fragment,” and “antibody derivative” as used herein, refer to aprotein fragment that comprises only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the terms antibody fragment or antigen-bindingfragment include: (i) the Fab fragment, having V_(L), C_(L), V_(H) andC_(H)1 domains; (ii) the Fab′ fragment, which is a Fab fragment havingone or more cysteine residues at the C-terminus of the C_(H)1 domain;(iii) the Fd fragment having V_(H) and C_(H)1 domains; (iv) the Fd′fragment having V_(H) and C_(H)1 domains and one or more cysteineresidues at the C-terminus of the CH1 domain; (v) the Fv fragment havingthe V_(L) and V_(H) domains of a single arm of an antibody; (vi) a dAbfragment (Ward et al., Nature 341, 544-546 (1989)) which consists of aV_(H) domain or a V_(L) domain; (vii) isolated CDR regions; (viii)F(ab′)₂ fragments, a bivalent fragment including two Fab′ fragmentslinked by a disulphide bridge at the hinge region; (ix) single chainantibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988));(x) “diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (see, e.g., EP 404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); (xi) “linear antibodies” comprising a pair of tandem Fdsegments (V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementarylight chain polypeptides, form a pair of antigen binding regions (Zapataet al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.5,641,870); and modified versions of any of the foregoing (e.g.,modified by the covalent attachment of polyalkylene glycol (e.g.,polyethylene glycol, polypropylene glycol, polybutylene glycol) or othersuitable polymer).

In some embodiments of the compositions and methods described herein, aROBO2 inhibitor or antagonist is a chimeric antibody derivative of aROBO2 antagonist antibody or antigen-binding fragment thereof.

The ROBO2 inhibitor or antagonist antibodies and antigen-bindingfragments thereof described herein can also be, in some embodiments, ahumanized antibody derivative.

In some embodiments, the ROBO2 inhibitor or antagonist antibodies andantigen-binding fragments thereof described herein include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the antibody, provided that the covalent attachment does notprevent the antibody from binding to the target antigen, e.g., ROBO2.

In some embodiments of the compositions and methods described herein,completely human antibodies are used, which are particularly desirablefor the therapeutic treatment of human patients.

In some embodiments of the compositions and methods described herein,the ROBO2 inhibitor comprises at least one antisense molecule capable ofblocking or decreasing the expression of a particular functional ROBO2by targeting nucleic acids encoding ROBO2, e.g., SEQ ID NO: 2 or SEQ IDNO: 4 or both, or relevant domains thereof. In some embodiments of thecompositions and methods described herein, the at least one antisensemolecule targets nucleic acids encoding the Ig SLIT binding domain ofROBO2. In some embodiments of the compositions and methods describedherein, the at least one antisense molecule targets nucleic acidsencoding the Ig1 SLIT binding domain of ROBO2 or both the Ig1 and Ig2SLIT binding domains of ROBO2. In some embodiments of the compositionsand methods described herein, the at least one antisense moleculetargets nucleic acids encoding the ROBO2 intracellular domain. In someembodiments of the compositions and methods described herein, the atleast one antisense molecule targets nucleic acids encoding the Nckintracellular binding domain comprising the four intracellular prolinerich motifs of ROBO2. Methods are known to those of ordinary skill inthe art for the preparation of antisense oligonucleotide molecules thatwill specifically bind ROBO2 mRNA without cross-reacting with otherpolynucleotides. Exemplary sites of targeting include, but are notlimited to, the initiation codon, the 5′ regulatory regions, includingpromoters or enhancers, the coding sequence, including any conservedconsensus regions, and the 3′ untranslated region. In some embodiment ofthese aspects and all such aspects described herein, the antisenseoligonucleotides are about 10 to about 100 nucleotides in length, about15 to about 50 nucleotides in length, about 18 to about 25 nucleotidesin length, or more. In certain embodiments, the antisenseoligonucleotides further comprise chemical modifications to increasenuclease resistance and the like, such as, for example, phosphorothioatelinkages and 2′-O-sugar modifications known to those of ordinary skillin the art.

In some embodiments of the compositions and methods described herein,the ROBO2 inhibitor comprises at least one short interfering RNA (siRNA)molecule capable of blocking or decreasing the expression of functionalROBO2 by targeting nucleic acids encoding or both isoforms of ROBO2,e.g., SEQ ID NO: 2 or SEQ ID NO: 4, or relevant domains thereof. In someembodiments of the compositions and methods described herein, the atleast one siRNA molecule targets nucleic acids encoding the Ig SLITbinding domain of ROBO2. In some embodiments of the compositions andmethods described herein, the at least one siRNA molecule targetsnucleic acids encoding the Ig1 SLIT binding domain of ROBO2 or both theIg1 and Ig2 SLIT binding domains of ROBO2. In some embodiments of thecompositions and methods described herein, the at least one siRNAmolecule targets nucleic acids encoding the ROBO2 intracellular domain.In some embodiments of the compositions and methods described herein,the at least one siRNA molecule targets nucleic acids encoding the Nckintracellular binding domain comprising the four intracellular prolinerich motifs of ROBO2. It is routine to prepare siRNA molecules that willspecifically target ROBO2 mRNA without cross-reacting with otherpolynucleotides. siRNA molecules for use in the compositions and methodsdescribed herein can be generated by methods known in the art, such asby typical solid phase oligonucleotide synthesis, and often willincorporate chemical modifications to increase half-life and/or efficacyof the siRNA agent, and/or to allow for a more robust deliveryformulation. Alternatively, siRNA molecules are delivered using a vectorencoding an expression cassette for intracellular transcription ofsiRNA.

In some embodiments of the compositions and methods described herein,the ROBO2 inhibitor is an RNA or DNA aptamer that binds to one or moreisoforms of ROBO2. In some embodiments of the compositions and methodsdescribed herein, a ROBO2 inhibitor or antagonist is an RNA or DNAaptamer that binds or physically interacts with ROBO2, and blocksinteractions between ROBO2 and a ligand or adaptor molecule, forexample, SLIT2 or Nck, respectively. In some embodiments of thecompositions and methods described herein, a ROBO-2 inhibitor orantagonist is an RNA or DNA aptamer that binds or physically interactswith ROBO2, and reduces, impedes, or blocks downstream ROBO2 signaling,such as SLIT2-ROBO-2 mediated inhibition of nephrin-mediated actinpolymerization, and/or elicitation of a cellular response to ROBO2. Insome embodiments of the compositions and methods described herein, theRNA or DNA aptamer binds to or physically interacts with the Ig SLITbinding domain of ROBO2. In some embodiments of the compositions andmethods described herein, the RNA or DNA aptamer binds to or physicallyinteracts with the Ig1SLIT binding domain of ROBO2 or both the Ig1 andIg2 SLIT binding domains of ROBO2, i.e., ROBO2(46-145) andROBO2(151-237) respectively of SEQ ID NO: 1, and ROBO2(30-129) andROBO2(135-221) respectively of SEQ ID NO: 3. In some embodiments of thecompositions and methods described herein, the RNA or DNA aptamer bindsto or physically interacts with the ROBO2 intracellular domain, i.e.,ROBO2(881-1378) of SEQ ID NO: 3. In some embodiments of the compositionsand methods described herein, the RNA or DNA aptamer binds to orphysically interacts with or blocks the Nck intracellular binding domaincomprising the four intracellular proline rich motifs of ROBO2.

In some embodiments of the compositions and methods described herein,the ROBO2 inhibitor is a small molecule compound or agent that targetsor binds to ROBO2, including, but not limited to, small peptides orpeptide-like molecules, soluble peptides, and synthetic non-peptidylorganic or inorganic compounds. As used herein, the term “smallmolecule” refers to a chemical agent which can include, but is notlimited to, a peptide, a peptidomimetic, an amino acid, an amino acidanalog, a polynucleotide, a polynucleotide analog, an aptamer, anucleotide, a nucleotide analog, an organic or inorganic compound (e.g.,including heterorganic and organometallic compounds) having a molecularweight less than about 10,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 5,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 1,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 500 grams per mole, and salts, esters,and other pharmaceutically acceptable forms of such compounds. Exemplarysites of small molecule binding include, but are not limited to, theportion of ROBO2 that binds to SLIT2 or to the adaptor Nck, i.e., theIg1SLIT binding domain of ROBO2 or both the Ig1 and Ig2 SLIT bindingdomains of ROBO2, the ROBO2 intracellular domain or the Nckintracellular binding domain comprising the four intracellular prolinerich motifs of ROBO2.

In some embodiments of the compositions and methods described herein, aROBO2 inhibitor or antagonist comprises a small molecule that binds toROBO2 and inhibits ROBO2 biological activity.

In some embodiments of the compositions and methods described herein,the ROBO2 inhibitor or antagonist comprises at least one ROBO2structural analog, such as a dominant negative ROBO2 polypeptide. Theterm ROBO2 structural analogs, as used herein, refers to compounds thathave a similar three dimensional structure as part of that of ROBO2 andwhich bind to SLIT2 and/or to Nck under physiological conditions invitro or in vivo, wherein the binding at least partially inhibits aROBO2 biological activity, such as SLIT2-ROBO2 mediated inhibition ofnephrin-mediated actin polymerization, and/or elicitation of a cellularresponse to ROBO2. Suitable ROBO2 structural analogs can be designed andsynthesized through molecular modeling of ROBO2-SLIT2 binding, forexample. The ROBO2 structural analogs can be monomers, dimers, or higherorder multimers in any desired combination of the same or differentstructures to obtain improved affinities and biological effects.

In some embodiments of the compositions and methods described herein, aROBO2 inhibitor or antagonist comprises at least one soluble ROBO2receptor or fusion polypeptide thereof, such as, for example, a ROBO2inhibitory polypeptide. In some such embodiments, the ROBO2 inhibitorypolypeptide is a dominant negative ROBO2 fusion protein. In someembodiments of the compositions and methods described herein, the ROBO2inhibitory polypeptide comprises the ROBO2 extracellular domain, forexample, the Ig1SLIT binding domain of ROBO2 or both the Ig1 and Ig2SLIT binding domains of ROBO2, with no intracellular ROBO2 domains.

ROBO2 inhibitors or antagonists for use in the compositions and methodsdescribed herein can be identified or characterized using methods knownin the art, such as protein-protein binding assays, biochemicalscreening assays, immunoassays, and cell-based assays, which are wellknown in the art, including, but not limited to, those described hereinin the Examples.

For example, to identify a molecule that inhibits interaction betweenROBO2 and its ligand, e.g., SLIT2, binding assays can be used. Forexample, ROBO2 or SLIT is immobilized on a microtiter plate by covalentor non-covalent attachment. The assay is performed by adding thenon-immobilized component (ligand or receptor), which can be labeled bya detectable label, to the immobilized component, in the presence orabsence of a test agent. When the reaction is complete, the non-reactedcomponents are removed and binding complexes are detected. If formationof binding complexes is inhibited by the presence of the test agent, thetest agent can be deemed a candidate antagonist that inhibits bindingbetween ROBO2 and SLIT2, for example. Cell-based or membrane-basedassays can also be used to identify ROBO2 inhibitors. In otherembodiments, by detecting and/or measuring levels of ROBO2 geneexpression, ROBO2 inhibitor molecules that inhibit ROBO2 gene expressioncan be tested. ROBO2 gene expression can be detected and/or measured bya variety of methods, such as real time RT-PCR, enzyme-linkedimmunosorbent assay (“ELISA”), Northern blotting, or flow cytometry, andas known to one of ordinary skill in the art.

Such identified ROBO2 inhibitors can further be tested using in vivoanimal models of chronic kidney disease, such as glomerular andinterstitial injury models (e.g., animal models of lupus nephritis,including mice of the NZB, (NZB×NZW) F1 hybrid (termed NZB/W), andcongenic derivatives thereof, MRL/lpr and BXSB strains), animal modelsof aging (e.g., aged Sprague Dawley rats and aged C57BL/6 mice);spontaneously hypertensive rats (SHR); Buffalo/mna rats, which are amodel of human idiopathic nephrotic syndrome; Munich Wistar Fromter(MWF) rats, which are a genetic model related to a congenital deficit innephron number being predisposed to the development of hypertension andsalt sensitivity in adulthood; primary podocyte-specific genetic FSGSmodels; HIV-associated nephropathy (HIVAN) transgenic mouse models;animal models of Alport syndrome, which comprise mutations of the α3,α4, or α5 chains of type IV collagen (COL4A3, COL4A4, and COL4A5);immune-induced models, such as the Thy-1 nephritis model, which is anexperimental rat model of mesangioproliferative glomerulonephritis(MsPGN), anti-glomerular basement membrane (GBM) models; and non-immuneinduced models.

As used herein, in regard to a ROBO2 inhibitor, “selectively binds” or“specifically binds” or “specific for” refers to the ability of a ROBO2inhibitor as described herein, such as, for example, a ROBO2 antagonistantibody or ROBO2 antigen-binding fragment thereof, to bind to a target,i.e., ROBO2, with a K_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M orless, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less,10⁻¹¹ M or less, or 10⁻¹² M or less. For example, if a ROBO2inhibitor/antagonist described herein binds to ROBO2 with a K_(D) of10⁻⁵ M or lower, but not to a related molecule, such as, for example,other ROBO family members, then the agent is said to specifically bindROBO2. Specific binding can be influenced by, for example, the affinityand avidity of, for example, the ROBO2 inhibitor/antagonist antibody orantigen-binding fragment thereof and the concentration of polypeptideagent. The person of ordinary skill in the art can determine appropriateconditions under which the polypeptide agents described hereinselectively bind the targets using any suitable methods, such astitration of a polypeptide agent in a suitable cell binding assay.

In regard to the methods of treating chronic kidney disease byinhibiting ROBO2 activity, the term “chronic kidney disease” or CKDrefers to renal diseases that slowly and progressively worsen over timedue to the progressive loss of nephrons and consequent loss of renalfunction. In the early stages, there may be no symptoms. The loss offunction usually takes months or years to occur. It may be so slow thatsymptoms do not appear until kidney function is less than one-tenth ofnormal. The final stage of chronic kidney disease is called end-stagerenal disease (ESRD). At this stage, the kidneys are no longer able toremove enough wastes and excess fluids from the body. The patient needsdialysis or a kidney transplant. Diabetes, which leads to diabeticnephropathy, and high blood pressure are the two most common causes ofchronic kidney disease and account for most cases. Other diseases andconditions that can damage the kidneys and lead to chronic kidneydisease, include: autoimmune disorders (such as systemic lupuserythematosus and scleroderma); birth defects of the kidneys (such aspolycystic kidney disease); certain toxic chemicals; glomerulonephritis;injury or trauma; kidney stones and infection; problems with thearteries leading to or inside the kidneys; some pain medications andother drugs (such as cancer drugs); reflux nephropathy (in which thekidneys are damaged by the backward flow of urine into the kidneys);etc. As used herein, “proteinuria” refers to the presence of an excessof serum proteins in the urine. Proteinuria can, in some embodiments, beindicative of kidney disease, but, by itself, is not conclusive.

Accordingly, in some embodiments of these aspects and all such aspectsdescribed herein, the subject having or at risk for a chronic kidneydisease has diabetic nephropathy.

By “reduce” or “inhibit” in terms of the chronic kidney disease andproteinuria treatment methods described herein is meant the ability tocause an overall decrease preferably of 20% or greater, 30% or greater,40% or greater, 45% or greater, more preferably of 50% or greater, of55% or greater, of 60% or greater, of 65% or greater, of 70% or greater,and most preferably of 75% or greater, 80% or greater, 85% or greater,90% or greater, or 95% or greater, for a given parameter or symptom of achronic kidney disease. Reduce or inhibit can refer to, for example,symptoms of the disorder being treated, for example, high bloodpressure, protein in the urine, etc.

High blood pressure is almost always present during all stages ofchronic kidney disease. A nervous system exam may show signs of nervedamage. The health care provider may hear abnormal heart or lung soundswhen listening with a stethoscope. The early symptoms of chronic kidneydisease are also symptoms of other illnesses. These symptoms can be theonly signs of kidney disease until the condition is more advanced.Symptoms of chronic kidney disease can include: appetite loss; generalill feeling and fatigue; headaches; itching (pruritus) and dry skin;nausea; weight loss without trying to lose weight; etc. Other symptomsthat can develop, especially when kidney function has gotten worse,include: abnormally dark or light skin; bone pain; brain and nervoussystem symptoms; drowsiness and confusion; problems concentrating orthinking; numbness in the hands, feet, or other areas; muscle twitchingor cramps; breath odor; easy bruising, bleeding, or blood in the stool;excessive thirst; frequent hiccups; low level of sexual interest andimpotence; stopping of menstrual periods (amenorrhea); shortness ofbreath; sleep problems, such as insomnia, restless leg syndrome, andobstructive sleep apnea; swelling of the feet and hands (edema);vomiting, typically in the morning.

Accordingly, in some embodiments of the methods described herein, aneffective amount of a composition comprising a ROBO2 inhibitor describedherein is administered to a subject in order to alleviate a symptom ofchronic kidney disease. As used herein, “alleviating a symptom chronickidney disease” is ameliorating any condition or symptom associated withthe chronic kidney disease. Alternatively, alleviating a symptom of achronic kidney disease can involve reducing one or more symptoms of thechronic kidney disease in the subject relative to an untreated controlsuffering from chronic kidney disease or relative to the subject priorto the treatment. As compared with an equivalent untreated control, orthe subject prior to the treatment with the ROBO2 inhibitor, suchreduction or degree of prevention is at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more, as measured by any standard technique.Desirably, the chronic kidney disease is significantly reduced orundetectable, as detected by any standard method known in the art, inwhich case the chronic kidney disease is considered to have beentreated. A patient who is being treated for a chronic kidney disease isone who a medical practitioner has diagnosed as having such a condition.Diagnosis can be by any suitable means known to one of ordinary skill inthe art. Diagnosis and monitoring can involve, for example, detectingthe level of specific proteins or molecules in a urine, blood, or serumsample, such as, for example, albumin, calcium, cholesterol, completeblood count (CBC), electrolytes, magnesium, phosphorous, potassium,sodium, or any combination thereof; assays to detect, for example,creatinine clearance; creatinine levels; BUN (blood urea nitrogen);through the use of specific techniques or procedures, such as anabdominal CT scan, abdominal MRI, abdominal ultrasound, kidney biopsy,kidney scan, kidney ultrasound; via detection of changes in results ofassays or tests for erythropoietin, PTH; bone density test, or VitaminD; or any combination of such detection methods and assays.

The terms “subject” and “individual” as used in regard to any of themethods described herein are used interchangeably herein, and refer toan animal, for example a human, recipient of the ROBO2 inhibitorsdescribed herein. For treatment of disease states which are specific fora specific animal such as a human subject, the term “subject” refers tothat specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike.

In some embodiments of these methods and all such methods describedherein, the method further comprises administering to the subject anadditional therapeutic agent, in addition to the ROBO2 inhibitor. Suchan additional therapeutic agent can be co-administered with the ROBO2inhibitor. As used herein, the phrase “co-administering” or to“co-administer” means the administration of a ROBO2 inhibitor describedherein and another compound, e.g., a therapeutic agent, separately,simultaneously, and/or sequentially over a period of time as determinedby a qualified care giver.

In some such embodiments, the additional therapeutic agent is anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin IIreceptor blocker (ARB), or a mineralocorticoid receptor (MR) antagonist.

ACE inhibitors for use with the ROBO2 inhibitors described hereininclude, but are not limited to, benazepril (marketed in the U.S. asLOTENSIN™), captopril (marketed in the U.S. as CAPOTEN™),enalapril/enalaprilat (marketed in the U.S. as VASOTEC™ oral andinjectable), fosinopril (marketed in the U.S. as MONOPRIL™), lisinopril(marketed in the U.S. as ZESTRIL™ and PRINIVIL™), moexipril (marketed inthe U.S. as UNIVASC™), perindopril (marketed in the U.S. as ACEON™),quinapril (marketed in the U.S. as ACCUPRIL™), ramipril (marketed in theU.S. as ALTACE™), and trandolapril (marketed in the U.S. as MAVIK™).ARBs for use with the ROBO2 inhibitors described herein includecandesartan (marketed in the U.S. as ATACAND™), irbesartan (marketed inthe U.S. as AVAPRO™), olmesartan (marketed in the U.S. as BENICAR™),losartan (marketed in the U.S. as COZAAR™), valsartan (marketed in theU.S. as DIOVAN™), telmisartan (marketed in the U.S. as MICARDIS™), andeprosartan (marketed in the U.S. as TEVETEN™).

In some embodiments of these methods and all such methods describedherein, the method further comprises administering to the subject aneffective amount of a diuretic, in addition to the ROBO2 inhibitor.Diuretics include, but are not limited to, torsemide (marketed in theU.S. as DEMADEX™), furosemide (marketed in the U.S. as LASIX™),bumetanide (marketed in the U.S. as BUMEX™), ethacrynic acid (marketedin the U.S. as EDECRIN™), torsemide (marketed in the U.S. as DEMADEX™),amiloride, (marketed in the U.S. as MIDAMOR™), acetazolamide (marketedin the U.S. as DIAMOX™), pamabrom (marketed in the U.S. as AQUA-BAN™),mannitol (marketed in the U.S. as ARIDOL™ or OSMITROL™), traimterene(marketed in the U.S. as DYRENIUM™), spironolactone (marketed in theU.S. as ALDACTONE™), amiloride (marketed in the U.S. as MIDAMOR™),indapamide (marketed in the U.S. as LOZOL™), hydrochlorothiazide(marketed in the U.S. as HYDRODIURIL™), metolazone (marketed in the U.S.as ZAROXOLYN™ or MYKROX™), methylclothiazide (marketed in the U.S. asAQUATENSEN™ or ENDURON™), hydrocholorthiazide (marketed in the U.S. asAQUAZIDE H™ or ESIDRIX™ or MICROZIDE™), chlorothiazide (marketed in theU.S. as DIURIL™), bendroflumethiazide (marketed in the U.S. asNATURETIN™), polythiazide (marketed in the U.S. as RENESE™),hydroflumethiazide (marketed in the U.S. as SALURON™), andchlorthalidone (marketed in the U.S. as THALITONE™). For a completelisting also see, e.g., Physician's Desk Reference, 2012 Edition, PDRNetwork (2011).

As used herein, in regard to any of the compositions and methodscomprising ROBO-2 inhibitors or combination treatments thereof describedherein, the terms “treat,” “treatment,” “treating,” or “amelioration”refer to therapeutic treatments, wherein the object is to reverse,alleviate, ameliorate, inhibit, slow down or stop the progression orseverity of a condition associated with, a disease or disorder. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition, disease or disorder associated with a chronickidney disease, such as, but not limited to, diabetic nephropathy.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of at least slowing of progress or worsening of symptoms thatwould be expected in absence of treatment. Beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

The term “effective amount” as used herein refers to the amount of aROBO-2 inhibitor described herein, needed to alleviate at least one ormore symptom of the disease or disorder being treated, and relates to asufficient amount of pharmacological composition to provide the desiredeffect. The term “therapeutically effective amount” therefore refers toan amount of the ROBO-2 inhibitor described herein, using the methods asdisclosed herein, that is sufficient to provide a particular effect whenadministered to a typical subject. An effective amount as used hereinwould also include an amount sufficient to delay the development of asymptom of the disease, alter the course of a symptom disease (forexample but not limited to, slow the progression of a symptom of thedisease), or reverse a symptom of the disease. Thus, it is not possibleto specify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the ROBO-2 inhibitor described herein, which achieves ahalf-maximal inhibition of measured function or activity) as determinedin cell culture, or in an appropriate animal model. Levels in plasma canbe measured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay. The dosage can be determined by a physician and adjusted, asnecessary, to suit observed effects of the treatment. Depending on thetype and severity of the chronic kidney disease, about 1 μg/kg to 100mg/kg (e.g., 0.1-20 mg/kg) of a ROBO2 inhibitor described herein is aninitial candidate dosage range for administration to the subject,whether, for example, by one or more separate administrations, or bycontinuous infusion.

Modes of Administration

The ROBO2 inhibitors or combination treatments thereof described hereincan be administered to a subject in need thereof by any appropriateroute which results in an effective treatment in the subject. As usedherein, the terms “administering,” and “introducing” are usedinterchangeably and refer to the placement of a ROBO-2 inhibitor into asubject by a method or route which results in at least partiallocalization of such agents at a desired site, such that a desiredeffect(s) is produced.

In some embodiments, the ROBO2 inhibitor is administered to a subjecthaving a chronic kidney disease by any mode of administration thatdelivers the agent systemically or to a desired surface or target, andcan include, but is not limited to, injection, infusion, instillation,and inhalation administration. To the extent that polypeptide agents canbe protected from inactivation in the gut, oral administration forms arealso contemplated. “Injection” includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments,the ROBO-2 inhibitors for use in the methods described herein areadministered by intravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally”as used herein, refer to modes of administration other than enteral andtopical administration, usually by injection. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer tothe administration of the ROBO-2 inhibitor, other than directly into atarget site, tissue, or organ, such as a tumor site, such that it entersthe subject's circulatory system and, thus, is subject to metabolism andother like processes.

For the clinical use of the methods described herein, administration ofthe ROBO-2 inhibitors described herein, can include formulation intopharmaceutical compositions or pharmaceutical formulations forparenteral administration, e.g., intravenous; mucosal, e.g., intranasal;ocular, or other mode of administration. In some embodiments, the ROBO-2inhibitors described herein can be administered along with anypharmaceutically acceptable carrier compound, material, or compositionwhich results in an effective treatment in the subject. Thus, apharmaceutical formulation for use in the methods described herein cancontain a ROBO-2 inhibitor, as described herein, in combination with oneor more pharmaceutically acceptable ingredients.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically acceptablecarrier” as used herein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, media, encapsulating material, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in maintaining thestability, solubility, or activity of, a ROBO-2 inhibitor. Each carriermust be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such ascocoa butter and suppository waxes; (8) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (9) glycols, such as propylene glycol; (10) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11)esters, such as ethyl oleate and ethyl laurate; (12) agar; (13)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17)Ringer's solution; (19) pH buffered solutions; (20) polyesters,polycarbonates and/or polyanhydrides; (21) bulking agents, such aspolypeptides and amino acids (22) serum components, such as serumalbumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24)other non-toxic compatible substances employed in pharmaceuticalformulations. Release agents, coating agents, preservatives, andantioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

The ROBO-2 inhibitors described herein can be specially formulated foradministration of the compound to a subject in solid, liquid or gelform, including those adapted for the following: (1) parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; (2) topical application, for example,as a cream, ointment, or a controlled-release patch or spray applied tothe skin; (3) intravaginally or intrarectally, for example, as apessary, cream or foam; (4) ocularly; (5) transdermally; (6)transmucosally; or (79) nasally. Additionally, a ROBO-2 inhibitor can beimplanted into a patient or injected using a drug delivery system. See,for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960.

Further embodiments of the formulations and modes of administration ofthe compositions comprising the ROBO-2 inhibitors described herein, thatcan be used in the methods described herein are described below.

Parenteral Dosage Forms.

Parenteral dosage forms of the ROBO-2 inhibitors can also beadministered to a subject with a chronic kidney condition by variousroutes, including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Sinceadministration of parenteral dosage forms typically bypasses thepatient's natural defenses against contaminants, parenteral dosage formsare preferably sterile or capable of being sterilized prior toadministration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection,controlled-release parenteral dosage forms, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe disclosure are well known to those skilled in the art. Examplesinclude, without limitation: sterile water; water for injection USP;saline solution; glucose solution; aqueous vehicles such as but notlimited to, sodium chloride injection, Ringer's injection, dextroseInjection, dextrose and sodium chloride injection, and lactated Ringer'sinjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and propylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

In some embodiments, compositions comprising an effective amount of aROBO2 inhibitor are formulated to be suitable for oral administration,for example as discrete dosage forms, such as, but not limited to,tablets (including without limitation scored or coated tablets), pills,caplets, capsules, chewable tablets, powder packets, cachets, troches,wafers, aerosol sprays, or liquids, such as but not limited to, syrups,elixirs, solutions or suspensions in an aqueous liquid, a non-aqueousliquid, an oil-in-water emulsion, or a water-in-oil emulsion. Suchcompositions contain a predetermined amount of the pharmaceuticallyacceptable salt of the disclosed compounds, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton, Pa. (1990).

Due to their ease of administration, tablets and capsules represent themost advantageous solid oral dosage unit forms, in which case solidpharmaceutical excipients are used. If desired, tablets can be coated bystandard aqueous or nonaqueous techniques. These dosage forms can beprepared by any of the methods of pharmacy. In general, pharmaceuticalcompositions and dosage forms are prepared by uniformly and intimatelyadmixing the active ingredient(s) with liquid carriers, finely dividedsolid carriers, or both, and then shaping the product into the desiredpresentation if necessary. In some embodiments, oral dosage forms arenot used for the antibiotic agent.

Typical oral dosage forms of the compositions comprising an effectiveamount of a ROBO2 inhibitor are prepared by combining thepharmaceutically acceptable salt of the ROBO2 inhibitor in an intimateadmixture with at least one excipient according to conventionalpharmaceutical compounding techniques. Excipients can take a widevariety of forms depending on the form of the composition desired foradministration. For example, excipients suitable for use in oral liquidor aerosol dosage forms include, but are not limited to, water, glycols,oils, alcohols, flavoring agents, preservatives, and coloring agents.Examples of excipients suitable for use in solid oral dosage forms(e.g., powders, tablets, capsules, and caplets) include, but are notlimited to, starches, sugars, microcrystalline cellulose, kaolin,diluents, granulating agents, lubricants, binders, and disintegratingagents.

Binders suitable for use in the pharmaceutical formulations describedherein include, but are not limited to, corn starch, potato starch, orother starches, gelatin, natural and synthetic gums such as acacia,sodium alginate, alginic acid, other alginates, powdered tragacanth,guar gum, cellulose and its derivatives (e.g., ethyl cellulose,cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinizedstarch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical formulationsdescribed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof. The binder orfiller in pharmaceutical compositions described herein is typicallypresent in from about 50 to about 99 weight percent of thepharmaceutical composition.

Disintegrants are used in the oral pharmaceutical formulations describedherein to provide tablets that disintegrate when exposed to an aqueousenvironment. A sufficient amount of disintegrant that is neither toolittle nor too much to detrimentally alter the release of the activeingredient(s) should be used to form solid oral dosage forms of theROBO2 inhibitors described herein. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Disintegrants that can be used toform oral pharmaceutical formulations include, but are not limited to,agar, alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used to form oral pharmaceutical formulations ofthe ROBO2 inhibitors described herein, include, but are not limited to,calcium stearate, magnesium stearate, mineral oil, light mineral oil,glycerin, sorbitol, mannitol, polyethylene glycol, other glycols,stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil,corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate,agar, and mixtures thereof. Additional lubricants include, for example,a syloid silica gel (AEROSIL® 200, manufactured by W. R. Grace Co. ofBaltimore, Md.), a coagulated aerosol of synthetic silica (marketed byDegussa Co. of Piano, Tex.), CAB-O-SIL® (a pyrogenic silicon dioxideproduct sold by Cabot Co. of Boston, Mass.), and mixtures thereof. Ifused at all, lubricants are typically used in an amount of less thanabout 1 weight percent of the pharmaceutical compositions or dosageforms into which they are incorporated.

In other embodiments, lactose-free pharmaceutical formulations anddosage forms are provided, wherein such compositions preferably containlittle, if any, lactose or other mono- or di-saccharides. As usedherein, the term “lactose-free” means that the amount of lactosepresent, if any, is insufficient to substantially increase thedegradation rate of an active ingredient. Lactose-free compositions ofthe disclosure can comprise excipients which are well known in the artand are listed in the USP (XXI)/NF (XVI), which is incorporated hereinby reference.

The oral formulations of the ROBO2 inhibitors further encompass, in someembodiments, anhydrous pharmaceutical compositions and dosage formscomprising the ROBO2 inhibitors described herein as active ingredients,since water can facilitate the degradation of some compounds. Forexample, the addition of water (e.g., 5%) is widely accepted in thepharmaceutical arts as a means of simulating long-term storage in orderto determine characteristics such as shelf life or the stability offormulations over time. See, e.g., Jens T. Carstensen, Drug Stability:Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995).Anhydrous pharmaceutical compositions and dosage forms described hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. Anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials)with or without desiccants, blister packs, and strip packs.

Aerosol Formulations.

A ROBO-2 inhibitor can be packaged in a pressurized aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. A ROBO-2inhibitor can also be administered in a non-pressurized form such as ina nebulizer or atomizer. A ROBO-2 inhibitor can also be administereddirectly to the airways in the form of a dry powder, for example, by useof an inhaler.

Suitable powder compositions include, by way of illustration, powderedpreparations of a ROBO-2 inhibitor, thoroughly intermixed with lactose,or other inert powders acceptable for intrabronchial administration. Thepowder compositions can be administered via an aerosol dispenser orencased in a breakable capsule which can be inserted by the subject intoa device that punctures the capsule and blows the powder out in a steadystream suitable for inhalation. The compositions can includepropellants, surfactants, and co-solvents and can be filled intoconventional aerosol containers that are closed by a suitable meteringvalve.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev.Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S, and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

The formulations of the ROBO-2 inhibitors described herein furtherencompass anhydrous pharmaceutical compositions and dosage formscomprising the disclosed compounds as active ingredients, since watercan facilitate the degradation of some compounds. For example, theaddition of water (e.g., 5%) is widely accepted in the pharmaceuticalarts as a means of simulating long-term storage in order to determinecharacteristics such as shelf life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Anhydrouspharmaceutical compositions and dosage forms of the disclosure can beprepared using anhydrous or low moisture containing ingredients and lowmoisture or low humidity conditions. Pharmaceutical compositions anddosage forms that comprise lactose and at least one active ingredientthat comprises a primary or secondary amine are preferably anhydrous ifsubstantial contact with moisture and/or humidity during manufacturing,packaging, and/or storage is expected. Anhydrous compositions arepreferably packaged using materials known to prevent exposure to watersuch that they can be included in suitable formulary kits. Examples ofsuitable packaging include, but are not limited to, hermetically sealedfoils, plastics, unit dose containers (e.g., vials) with or withoutdesiccants, blister packs, and strip packs.

Controlled and Delayed Release Dosage Forms.

In some embodiments of the aspects described herein, a ROBO-2 inhibitorcan be administered to a subject by controlled- or delayed-releasemeans. Ideally, the use of an optimally designed controlled-releasepreparation in medical treatment is characterized by a minimum of drugsubstance being employed to cure or control the condition in a minimumamount of time. Advantages of controlled-release formulationsinclude: 1) extended activity of the drug; 2) reduced dosage frequency;3) increased patient compliance; 4) usage of less total drug; 5)reduction in local or systemic side effects; 6) minimization of drugaccumulation; 7) reduction in blood level fluctuations; 8) improvementin efficacy of treatment; 9) reduction of potentiation or loss of drugactivity; and 10) improvement in speed of control of diseases orconditions. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2(Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-releaseformulations can be used to control a compound of formula (I)'s onset ofaction, duration of action, plasma levels within the therapeutic window,and peak blood levels. In particular, controlled- or extended-releasedosage forms or formulations can be used to ensure that the maximumeffectiveness of a compound of formula (I) is achieved while minimizingpotential adverse effects and safety concerns, which can occur both fromunder-dosing a drug (i.e., going below the minimum therapeutic levels)as well as exceeding the toxicity level for the drug.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the ROBO-2inhibitors described herein. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each ofwhich is incorporated herein by reference in their entireties. Thesedosage forms can be used to provide slow or controlled-release of one ormore active ingredients using, for example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)),multilayer coatings, microparticles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Additionally, ion exchange materials can be used to prepareimmobilized, adsorbed salt forms of the disclosed compounds and thuseffect controlled delivery of the drug. Examples of specific anionexchangers include, but are not limited to, DUOLITE® A568 and DUOLITE®AP143 (Rohm & Haas, Spring House, Pa. USA).

In some embodiments of the methods described herein, a ROBO-2 inhibitorfor use in the methods described herein is administered to a subject bysustained release or in pulses. Pulse therapy is not a form ofdiscontinuous administration of the same amount of a composition overtime, but comprises administration of the same dose of the compositionat a reduced frequency or administration of reduced doses. Sustainedrelease or pulse administrations are particularly preferred when thedisorder occurs continuously in the subject, for example where thesubject has chronic kidney disease. Each pulse dose can be reduced andthe total amount of a ROBO-2 inhibitor described herein administeredover the course of treatment to the subject or patient is minimized.

The interval between pulses, when necessary, can be determined by one ofordinary skill in the art. Often, the interval between pulses can becalculated by administering another dose of the composition when thecomposition or the active component of the composition is no longerdetectable in the subject prior to delivery of the next pulse. Intervalscan also be calculated from the in vivo half-life of the composition.Intervals can be calculated as greater than the in vivo half-life, or 2,3, 4, 5 and even 10 times greater the composition half-life. Variousmethods and apparatus for pulsing compositions by infusion or otherforms of delivery to the patient are disclosed in U.S. Pat. Nos.4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.

In some embodiments, sustained-release preparations comprising theROBO-2 inhibitor can be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the inhibitor, in which matrices are in the form ofshaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations comprising the ROBO-2 inhibitors to be used for in vivoadministration are preferably sterile. This is readily accomplished byfiltration through, for example, sterile filtration membranes, and othermethods known to one of skill in the art.

Also provided herein, in some aspects, are assays, methods, and systemsfor determining whether an individual has a chronic kidney disease or apre-disposition for a chronic kidney disease or proteinuria based onexpression profiles or sequence information of ROBO2 as a biomarkerindicative of chronic kidney disease or a pre-disposition for a chronickidney disease or proteinuria. As demonstrated herein, ROBO2 is usefulas a biomarker to identify a subject having chronic kidney disease or athigh risk for chronic kidney disease or proteinuria or to monitor theeffects of treatment on the progression of chronic kidney disease orproteinuria.

As used herein, a “biomarker” refers to an organic biomolecule which isdifferentially present in a sample taken from a subject of onephenotypic status (e.g., having a disease) as compared with anotherphenotypic status (e.g., not having the disease). A biomarker isdifferentially present between different phenotypic statuses if the meanor median expression level of the biomarker in the different groups iscalculated to be statistically significant. Common tests for statisticalsignificance include, among others, t-test, ANOVA, Kruskal-Wallis,Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or incombination, provide measures of relative risk that a-subject belongs toone phenotypic status or another. As such, they are useful as markersfor disease (diagnostics), therapeutic effectiveness of a drug(theranostics) and of drug toxicity.

ROBO2 expression for use in the assays described herein can be detectedby any suitable method, including detection or protein levels ordetection of mRNA expression levels. ROBO2 polypeptide can be detectedin any form that may be found in a biological sample obtained from asubject, or in any form that may result from manipulation of thebiological sample (e.g., as a result of sample processing). Modifiedforms of ROBO2 can include modified proteins that are a product ofallelic variants, splice variants, post-translational modification(e.g., glycosylation, proteolytic cleavage (e.g., fragments of a parentprotein), glycosylation, phosphorylation, lipidation, oxidation,methylation, cysteinylation, sulphonation, acetylation, and the like),oligomerization, de-oligomerization (to separate monomers from amultimeric form of the protein), denaturation, and the like.

The assays described herein can be designed to detect all forms orparticular forms of ROBO2. Where desired, differentiation betweendifferent forms of ROBO2, e.g., different isoforms, can be accomplishedby use of detection methods dependent upon physical characteristics thatdiffer between the forms, e.g., different molecular weight, differentmolecular size, presence/absence of different epitopes, and the like.

Accordingly, provided herein, in some aspects, are assays for thediagnosis of a subject at having chronic kidney disease or at risk forchronic kidney disease or proteinuria, the assay comprising: measuringthe level of ROBO2 protein or nucleic acid in a biological sampleobtained from the subject, wherein if the level of the ROBO2 in thebiological sample from the subject is at the same level or greater than(e.g., greater than by a statistically significant amount) a thresholdreference level for ROBO2, the subject likely is at risk for chronickidney disease or proteinuria or has chronic kidney disease. Forexample, an increase in the level of ROBO2 by more than about 10%, ormore than about 20%, or more than about 30%, or more than about 40%, ormore than about 50%, or more than about 60%, or more, as compared to areference threshold level of ROBO2. In some embodiments, the increase inthe level of ROBO2 is at least one standard deviation greater than, orat least two standard deviations, or more, greater than a median or meanROBO2 reference threshold level. Such median or mean ROBO2 referencelevels can be obtained, for example, from five or more samples obtainedfrom subjects not having chronic kidney disease or proteinuria, or fromfive or more samples obtained from the same subject at differenttimepoints.

In some embodiments of these assays, the amount of ROBO2 measured in abiological sample is compared to a reference threshold level, or areference biological sample, such as biological sample obtained from anage-matched normal control (e.g., an age-matched subject not having arisk of chronic kidney disease or proteinuria), or a healthy subject,e.g., a healthy individual.

In some embodiments, the assays, systems and kits as disclosed hereinare also useful for monitoring a course of treatment being administeredto a subject. For example, one can measure the level of ROBO2 in abiological sample in the subject at a first timepoint (e.g., t1) andcompare with the ROBO2 biomarker reference threshold level, and if themeasured level for ROBO2 is the same or higher than the referencethreshold level, the subject can be administered an appropriatetherapeutic treatment or regimen to delay or reduce the occurrence ofchronic kidney disease or proteinuria, e.g., for example, increaseexercise, reduce heart pressure, adjust diet etc. as disclosed in themethods herein, and then the level of the panel of ROBO2 biomarkerprotein can be measured at a second (e.g., t2) and subsequent timepoints(e.g., t3, t4, t5, t5 . . . etc.), and compared to levels of tROBO2 atone or more time points (e.g., at t1 or any subsequent timepoint) or thereference threshold levels of ROBO2 to determine if the therapeutictreatment or medical treatment or regimen for the treatment to reducethe risk of, delay, or reduce the occurrence of chronic kidney diseaseor proteinuria is effective. In some such embodiments, the assays,systems and kits as disclosed herein can be used to monitor atherapeutic treatment in a symptomatic subject (e.g., a subject known tohave chronic kidney disease or proteinuria), where an effectivetreatment can be a decrease in ROBO2 in the subject, or alternativelythe assays, systems and kits as disclosed herein can be used to monitorthe effect of prophylactic treatment in an asymptomatic subject (e.g.,to prevent chronic kidney disease or proteinuria occurring in asubject), for example, where the subject has been identified to be atrisk of chronic kidney disease or proteinuria according to the methodsas disclosed herein, or others known in the art, or due to hereditaryreasons, for example.

The term “sample” as used herein generally refers to any materialcontaining nucleic acid, either DNA or RNA, or amino acids. Generally,such material will be in the form of a blood sample, stool sample,tissue sample, cells, bacteria, histology section, or buccal swab.Samples can be prepared, for example samples can be fresh, fixed,frozen, or embedded in paraffin. The term “biological sample” as usedherein refers to a cell or population of cells or a quantity of tissueor fluid from a subject. Most often, the sample has been removed from asubject, but the term “biological sample” can also refer to cells ortissue analyzed in vivo, i.e. without removal from the subject. Often, a“biological sample” will contain cells from the animal, but the term canalso refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine, that can be used to measure geneexpression levels. Biological samples include, but are not limited to,tissue biopsies, scrapes, whole blood, plasma, serum, urine, saliva,cell culture, or cerebrospinal fluid. Biological samples also includetissue biopsies, cell culture. A biological sample or tissue sample canrefers to a sample of tissue or fluid isolated from an individual,including but not limited to, for example, urine, blood, plasma, serum,kidney biopsy, stool, sputum, spinal fluid, pleural fluid, nippleaspirates, lymph fluid, the external sections of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, milk, cells(including but not limited to blood cells), tumors, organs, and alsosamples of an in vitro cell culture constituent. In some embodiments,where a urine sample is obtained, the urine sample is centrifuged topellet any kidney cells, on which the assays and methods describedherein can be performed. In some embodiments, the sample is from akidney biopsy, such as a core needle biopsy of a kidney or portionthereof, such as a podocyte sample. In addition, fine needle aspiratesamples are used. In some embodiments, the biological samples can beprepared, for example biological samples can be fresh, fixed, frozen, orembedded in paraffin. The sample can be obtained by removing a sample ofcells from a subject, but can also be accomplished by using previouslyisolated cells (e.g. isolated by another person), or by performing themethods described herein in vivo.

The term “expression” as used herein refers to interchangeably to theexpression of a polypeptide or protein or expression of a polynucleotideor expression of a gene. Expression also refers to the expression ofpre-translational modified and post-translationally modified proteins,as well as expression of pre-mRNA molecules, alternatively spliced andmature mRNA molecules. Expression of a polynucleotide can be determined,for example, by measuring the production of RNA transcript molecules,for example messenger RNA (mRNA) transcript levels. Expression of aprotein or polypeptide can be determined, for example, by immunoassayusing an antibody(ies) that bind with the polypeptide. The term “encode”as it is applied to polynucleotides refers to a polynucleotide which issaid to “encode” a polypeptide or protein if, in its native state orwhen manipulated by methods well known to those skilled in the art, itcan be transcribed to produce the RNA which can be translated into anamino acid sequence to generate the polypeptide and/or a fragmentthereof. The antisense strand is the complement of such a nucleic acid,and the encoding sequence can be deduced therefrom. The term“endogenously expressed” or “endogenous expression” refers to theexpression of a gene product at normal levels and under normalregulation for that cell type.

Detection methods that can be used with the assays, methods, and systemsdescribed herein to measure levels of ROBO2 protein or nucleic acid in asample or biological sample include optical methods, electrochemicalmethods (voltametry and amperometry techniques), atomic forcemicroscopy, and radio frequency methods, e.g., multipolar resonancespectroscopy. Optical methods include microscopy, both confocal andnon-confocal, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry).

In those embodiments of the assays, methods, and systems describedherein in which the level of ROBO2 protein is determined, such as, forexample, the level of a protein of SEQ ID NO: 1 or SEQ ID NO: 3, one canuse any proteomic approach commonly known to persons of ordinary skillin the art to measure the level of biomarker proteins in a biologicalsample. The measurement can be either quantitative or qualitative, solong as the measurement is capable of determining or indicating whetherthe level of ROBO2 protein in the biological sample is the same as, orabove or below a reference threshold value for ROBO2 protein.

The measured level of ROBO2 protein can, in some embodiments, be aprimary measurement of the level of ROBO2 protein measuring the quantityof ROBO2 protein itself, such as by detecting the number of ROBO2protein molecules in the sample) or it can be, in some embodiments, asecondary measurement of ROBO2 protein (a measurement from which thequantity of ROBO2 protein can be but not necessarily deduced, such as ameasure of functional activity or a measure of nucleic acid, such asmRNA, encoding ROBO2 protein). Qualitative data can also be derived orobtained from primary measurements.

In some embodiments of the assays and methods described herein, ROBO2protein levels can be measured using an affinity-based measurementtechnology. “Affinity” as relates to an antibody is a term wellunderstood in the art and means the extent, or strength, of binding ofantibody to the binding partner, such as a biomarker as described herein(or epitope thereof). Affinity can be measured and/or expressed in anumber of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D) or Kd), apparent equilibriumdissociation constant (K_(D′) or K_(d′)), and IC₅₀ (amount needed toeffect 50% inhibition in a competition assay; used interchangeablyherein with “30”). It is understood that, for purposes of thisinvention, an affinity is an average affinity for a given population ofantibodies which bind to an epitope.

Affinity-based measurement technology utilizes a molecule thatspecifically binds to the biomarker protein being measured (an “affinityreagent,” such as an antibody or aptamer), although other technologies,such as spectroscopy-based technologies (e.g., matrix-assisted laserdesorption ionization-time of flight, MALDI-TOF spectroscopy) or assaysmeasuring bioactivity (e.g., assays measuring mitogenicity of growthfactors) can also be used. Affinity-based technologies for use with theassays and methods described herein can include antibody-based assays(immunoassays) and assays utilizing aptamers (nucleic acid moleculeswhich specifically bind to other molecules), such as ELONA.Additionally, assays utilizing both antibodies and aptamers are alsocontemplated (e.g., a sandwich format assay utilizing an antibody forcapture and an aptamer for detection). A wide variety of affinity-basedassays are also known in the art.

Affinity-based assays typically utilize at least one epitope derivedfrom the biomarker protein, i.e., ROBO2, and many affinity-based assayformats utilize more than one epitope (e.g., two or more epitopes areinvolved in “sandwich” format assays; at least one epitope is used tocapture the biomarker protein, and at least one different epitope isused to detect the marker).

Affinity-based assays can be in competition or direct reaction formats,utilize sandwich-type formats, and can further be heterogeneous (e.g.,utilize solid supports) or homogenous (e.g., take place in a singlephase) and/or utilize immunoprecipitation. Many assays involve the useof labeled affinity reagent (e.g., antibody, polypeptide, or aptamer);the labels can be, for example, enzymatic, fluorescent,chemiluminescent, radioactive, or dye molecules. Assays which amplifythe signals from the probe are also known; examples of which are assayswhich utilize biotin and avidin, and enzyme-labeled and mediatedimmunoassays, such as ELISA and ELONA assays. For example, the biomarkerconcentrations from biological fluid samples may be measured by LUMINEX®assay or ELISA. Either of the biomarker or reagent specific for thebiomarker can be attached to a surface and levels can be measureddirectly or indirectly.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels can be measured using an immunoassayaffinity-based measurement technology.

Immunoassay technologies can include any immunoassay technology whichcan quantitatively or qualitatively measure the level of ROBO2 proteinin a biological sample. Suitable immunoassay technologies include, butare not limited to radioimmunoassay, ELISA (enzyme-linked immunosorbantassay), “sandwich” immunoassays, immunoradiometric assays,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blot analysis,immunoprecipitations, immunofluorescence assays, immunoelectrophoresisassays, fluoroimmunoassay (FiA), immunoradiometric assay (IRMA),immunoenzymometric assay (IEMA), immunoluminescence assay andimmunofluorescence assay (Madersbacher S, Berger P. Antibodies andimmunoassays. Methods 2000; 21:41-50), chemiluminescent assay,immuno-PCR, and western blot assay. Likewise, aptamer-based assays whichcan quantitatively or qualitatively measure the level of a biomarker ina biological sample can be used in the assays, methods, and systemsdescribed herein. Generally, aptamers can be substituted for antibodiesin nearly all formats of immunoassay, although aptamers allow additionalassay formats (such as amplification of bound aptamers using nucleicacid amplification technology such as PCR (U.S. Pat. No. 4,683,202) orisothermal amplification with composite primers (U.S. Pat. Nos.6,251,639 and 6,692,918).

In some embodiments of the assays, methods, and systems describedherein, where ROBO2 protein levels are measured using an immunoassayaffinity-based measurement technology, the immunoassay is performed bymeasuring the extent of the protein/antibody interaction of thebiomarker/antibody interaction. Any known method of immunoassay can beused.

In some embodiments, a binding partner, e.g., an antibody or a ligandbinding to the ROBO2 protein in the binding assay, is preferably alabeled specific binding partner, but not necessarily an antibody. Thebinding partner is usually labeled itself, but alternatively it can bedetected by a secondary reaction in which a signal is generated, e.g.from another labeled substance.

Thus, the antibody which specifically binds to ROBO2 protein can be usedin the assays, methods, and systems described herein to determine thepresence and/or amount of ROBO2 protein n a biological sample, which canbe used to detect the increased or decreased concentration of ROBO2protein present in a diagnostic sample. Such antibodies can be raised byany of the methods well known in the immunodiagnostics field. Theantibodies can be anti-ROBO2 protein antibodies to any biologicallyrelevant state of the protein. Thus, for example, they could be raisedagainst the unglycosylated form of ROBO2 protein, which exists in thebody in a glycosylated form, or against a peptide carrying a relevantepitope of ROBO2 protein.

In some embodiments of these assays, method, and systems, an amplifiedassay form can be used, whereby an enhanced “signal” is produced from arelatively low level of protein to be detected. One particular form ofan amplified immunoassay is enhanced chemiluminescent assay. Forexample, the antibody is labeled with horseradish peroxidase, whichparticipates in a chemiluminescent reaction with luminol, a peroxidesubstrate and a compound which enhances the intensity and duration ofthe emitted light, typically 4-iodophenol or 4-hydroxycinnamic acid.

In some embodiments of these assays, method, and systems, an amplifiedimmunoassay can be used comprising immuno-PCR. In this technique, theantibody is covalently linked to a molecule of arbitrary DNA comprisingPCR primers, whereby the DNA with the antibody attached to it isamplified by the polymerase chain reaction. See E. R. Hendrickson etal., Nucleic Acids Research 23: 522-529 (1995).

Accordingly, in all embodiments of the assays, method, and systemsdescribed herein, the level of ROBO2 protein can be determined using aprotein-binding agent, also referred to herein as “protein-bindingentity” or an “affinity reagent” can be used, in particular, antibodies.For instance, the affinity reagents, in particular, antibodies such asanti-biomarker antibodies can be used in an immunoassay, particularly inan ELISA (Enzyme Linked Immunosorbent Assay). In embodiments where thelevel of a biomarker protein can be measured in a biological sampleusing methods commonly known in the art, and including, for example butnot limited to isoform-specific chemical or enzymatic cleavage ofisoform proteins, immunobloting, immunohistochemical analysis, ELISA,and mass spectrometry.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using “Enzyme-LinkedImmunosorbent Assay (ELISA).” ELISA is a technique for detecting andmeasuring the concentration of an antigen using a labeled (e.g. enzymelinked) form of the antibody. There are different forms of ELISA, whichare well known to those skilled in the art. The standard techniquesknown in the art for ELISA are described in “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc.,1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using a sandwich assay ELISA.In a “sandwich ELISA”, an antibody (e.g. anti-enzyme) is linked to asolid phase (i.e. a microtiter plate) and exposed to a biological samplecontaining antigen (e.g. enzyme). The solid phase is then washed toremove unbound antigen. A labeled antibody (e.g. enzyme linked) is thenbound to the bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Accordingly, using this method, afirst antibody to ROBO2 protein is bound to the solid phase such as awell of a plastics microtiter plate, and incubated with the sample andwith a labeled second antibody specific to ROBO2 protein to be assayed.Examples of enzymes that can be linked to the antibody are alkalinephosphatase, horseradish peroxidase, luciferase, urease, andB-galactosidase. The enzyme linked antibody reacts with a substrate togenerate a colored reaction product that can be measured.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using an antibody captureassay or competitive ELISA. In a “competitive ELISA”, antibody isincubated with a sample containing antigen (i.e. enzyme). Theantigen-antibody mixture is then contacted with a solid phase (e.g. amicrotiter plate) that is coated with antigen (i.e., enzyme). The moreantigen present in the sample, the less free antibody that will beavailable to bind to the solid phase. A labeled (e.g., enzyme linked)secondary antibody is then added to the solid phase to determine theamount of primary antibody bound to the solid phase. Accordingly, insome such embodiments, a biological test sample is allowed to bind to asolid phase, and the anti-ROBO2 protein antibody (e.g., antibodies thatspecifically bind ROBO2 protein) can be added and allowed to bind. Afterwashing away unbound material, the amount of antibody bound to the solidphase is determined using a labeled second antibody, anti- to the first.

In some embodiments of these assays, method, and systems, a label ispreferably an enzyme. The substrate for the enzyme can be, for example,color-forming, fluorescent or chemiluminescent.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using immunohistochemistry. Inan “immunohistochemistry assay” a section of tissue is tested forspecific proteins by exposing the tissue to antibodies that are specificfor the protein that is being assayed. The antibodies are thenvisualized by any of a number of methods to determine the presence andamount of the protein present. Examples of methods used to visualizeantibodies are, for example, through enzymes linked to the antibodies(e.g., luciferase, alkaline phosphatase, horseradish peroxidase, orbeta-galactosidase), or chemical methods (e.g., DAB/Substratechromagen). The sample is then analyzed microscopically, most preferablyby light microscopy of a sample stained with a stain that is detected inthe visible spectrum, using any of a variety of such staining methodsand reagents known to those skilled in the art.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using radioimmunoassays. Aradioimmunoassay is a technique for detecting and measuring theconcentration of an antigen, i.e., ROBO2, using a labeled (e.g.radioactively or fluorescently labeled) form of the antigen. Examples ofradioactive labels for antigens include 3H, 14C, and 125I. Theconcentration of ROBO2 in a biological sample is measured by having theROBO2 in the biological sample compete with the labeled (e.g.radioactively) ROBO2 for binding to an antibody to ROBO2. To ensurecompetitive binding between the labeled ROBO2 and the unlabeled ROBO2,the labeled ROBO2 is present in a concentration sufficient to saturatethe binding sites of the antibody. The higher the concentration of ROBO2in the sample, the lower the concentration of labeled ROBO2 that willbind to the antibody.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using an immunoradiometricassay (IRMA). IRMA is an immunoassay in which the antibody reagent isradioactively labeled. An IRMA requires the production of a multivalentantigen conjugate, by techniques such as conjugation to a protein e.g.,rabbit serum albumin (RSA). The multivalent antigen conjugate must haveat least 2 antigen residues per molecule and the antigen residues mustbe of sufficient distance apart to allow binding by at least twoantibodies to the antigen. For example, in an IRMA the multivalentantigen conjugate can be attached to a solid surface such as a plasticsphere. Unlabeled “sample” antigen and antibody to antigen which isradioactively labeled are added to a test tube containing themultivalent antigen conjugate coated sphere. The antigen in the samplecompetes with the multivalent antigen conjugate for antigen antibodybinding sites. After an appropriate incubation period, the unboundreactants are removed by washing and the amount of radioactivity on thesolid phase is determined. The amount of bound radioactive antibody isinversely proportional to the concentration of antigen in the sample.

Other techniques can be used to detect the level of ROBO2 protein in abiological sample can be performed according to a practitioner'spreference, and based upon the present disclosure and the type ofbiological sample (i.e. plasma, urine, tissue sample etc.). One suchtechnique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Detectably labeled anti-ROBO2 antibodies orprotein binding molecules can then be used to assess the level of ROBO2protein, where the intensity of the signal from the detectable labelcorresponds to the amount of ROBO2 protein. Levels of the amount ofROBO2 protein present can also be quantified, for example bydensitometry.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using mass spectrometry suchas MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-massspectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), highperformance liquid chromatography-mass spectrometry (HPLC-MS), capillaryelectrophoresis-mass spectrometry, nuclear magnetic resonancespectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS,ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos:20030199001, 20030134304, 20030077616, which are incorporated herein intheir entirety by reference.

In some such embodiments, these methodologies can be combined with themachines, computer systems and media to produce an automated system fordetermining the level of ROBO2 protein in a biological sample andanalysis to produce a printable report which identifies, for example,the level of ROBO2 protein in a biological sample. In some instances,the measurement of the level of ROBO2 is done remotely from thedetermination and comparison modules.

Mass spectrometry methods are well known in the art and have been usedto quantify and/or identify biomolecules, such as proteins (see, e.g.,Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20:383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8:393-400). Further, mass spectrometric techniques have been developedthat permit at least partial de novo sequencing of isolated proteins.Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad.Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).

In certain embodiments, a gas phase ion spectrophotometer is used. Inother embodiments, laser-desorption/ionization mass spectrometry is usedto analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937(Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait) whichare incorporated herein by reference.

In SELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe protein of interest. In another variant, the surface is derivatizedwith energy absorbing molecules that are not desorbed when struck withthe laser. In another variant, the surface is derivatized with moleculesthat bind the protein of interest and that contain a photolytic bondthat is broken upon application of the laser. In each of these methods,the derivatizing agent generally is localized to a specific location onthe substrate surface where the sample is applied. See, e.g., U.S. Pat.No. 5,719,060 and WO 98/59361 which are incorporated herein byreference. The two methods can be combined by, for example, using aSELDI affinity surface to capture an analyte and addingmatrix-containing liquid to the captured analyte to provide the energyabsorbing material.

For additional information regarding mass spectrometers, see, e.g.,Principles of Instrumental Analysis, 3rd edition, Skoog, SaundersCollege Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia ofChemical Technology, 4th ed. Vol. 15 (John Wiley & Sons, New York 1995),pp. 1071-1094.

Detection of the level of ROBO2 protein will typically depend on thedetection of signal intensity. This, in turn, can reflect the quantityand character of a polypeptide bound to the substrate. For example, incertain embodiments, the signal strength of peak values from spectra ofa first sample and a second sample can be compared (e.g., visually, bycomputer analysis etc.), to determine the relative amounts of particularbiomolecules. Software programs such as the Biomarker Wizard program(Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid inanalyzing mass spectra. The mass spectrometers and their techniques arewell known to those of skill in the art.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using gel electrophoresistechniques, in particular SDS-PAGE (Sodium Dodecylsulfate PolyacrylamideGel Elektrophoresis), especially two dimensional PAGE (2D-PAGE),preferably two dimensional SDS-PAGE (2D-SDS-PAGE). According to aparticular example, the assay is based on 2D-PAGE, in particular, usingimmobilized pH gradients (IPGs) with a pH range preferably over pH 4-9.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using gel electrophoresistechniques, in particular, the above mentioned techniques combined withother protein separation methods, particularly methods known to thoseskilled in the art, in particular, chromatography and/or size exclusion.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using resonance techniques, inparticular, plasma surface resonance.

In some embodiments of the assays, methods, and systems describedherein, ROBO2 protein levels are measured using a protein biochip. Abiochip generally comprises a solid substrate having a substantiallyplanar surface, to which a capture reagent (e.g., an adsorbent oraffinity reagent) is attached. Frequently, the surface of a biochipcomprises a plurality of addressable locations having bound capturereagent bound. The biochip may also include bound capture reagent thatserves as a control. Protein biochips are biochips adapted for thecapture of polypeptides. Many protein biochips are described in the art.These include, for example, protein biochips produced by CiphergenBiosystems, Inc. (Fremont, Calif.), Zyomyx (Hayward, Calif.), Invitrogen(Carlsbad, Calif.), Biacore (Uppsala, Sweden) and Procognia (Berkshire,UK). Examples of such protein biochips are described in the followingpatents or published patent applications: U.S. Pat. No. 6,225,047(Hutchens & Yip); U.S. Pat. No. 6,537,749 (Kuimelis and Wagner); U.S.Pat. No. 6,329,209 (Wagner et al.); PCT International Publication No. WO00156934 (Englert et al.); PCT International Publication No. WO031048768 (Boutell et al.) and U.S. Pat. No. 5,242,828 (Bergstrom etal.).

The reference threshold levels or values of ROBO2 protein levels usedfor comparison with the level of ROBO2 protein from a subject can vary,depending on the aspect or embodiment described herein being practiced,as will be understood throughout this specification, and below. Areference threshold value can be based on an individual sample value,such as for example, a value obtained from a biological sample from thesubject being tested, but at an earlier point in time (e.g., at a firsttimepoint (t1), e.g., a first biomarker level measured, or at a secondtimepoint (t2), e.g.,). A reference threshold value can also be based ona pool of samples, for example, value(s) obtained from samples from apool of subjects being tested. For example, in some embodiments,reference threshold values for ROBO2 protein are based on measured the50% value (e.g., median) of ROBO2 protein measured in subjects known tohave chronic kidney disease or proteinuria. For example, subjects in thetop 50% (e.g., at or above the median level) for ROBO2 protein can beselected to be at risk of having chronic kidney disease or proteinuria.Reference value(s) can also be based on a pool of samples including orexcluding the sample(s) to be tested. The reference value can be basedon a large number of samples, such as from population of healthysubjects of the chronological age-matched group, or from subjects who donot have or do not have a risk of chronic kidney disease or proteinuria.In some embodiments, the reference value can be at least one, moretypically at least two, standard deviations above the mean or median ofany of these assays or a predetermined mean or median.

For assessing the risk of a subject likely to experience or have chronickidney disease or proteinuria by the assays, methods, and systems asdisclosed herein, a “reference threshold value” is typically apredetermined reference threshold level, such as the median urine, serumor blood ROBO2 protein obtained from a population of healthy subjectsthat are in the chronological age group matched with the chronologicalage of the tested subject. As indicated earlier, in some situations, thereference samples can also be gender matched, and/or matched based onethnicity. In some embodiments, the reference threshold value for ROBO2protein is the median level for that biomarker in a type of biologicalsample, e.g., urine, blood, serum, in a panel subjects for the sameethnicity, e.g., Caucasian, Black, Hispanic, Asian, and Asian-Indian,Pakistani, Middle Eastern and/or Pacific Islander.

For assessing the risk of a subject likely to experience or have chronickidney disease or proteinuria by the assays, methods, and systems asdisclosed herein, the reference threshold level for ROBO2 protein can bea predetermined level, such as an average or median of levels obtainedfrom a population of healthy subjects that are in the chronological agegroup matched with the chronological age of the tested subject.Alternately, the reference threshold level for ROBO2 protein can be ahistorical reference level for the particular subject that was obtainedfrom a sample derived from the same subject, but at an earlier point intime, and/or when the subject did not have a risk of chronic kidneydisease or proteinuria. In some instances, the reference threshold levelfor ROBO2 protein can be a historical reference level of ROBO2 proteinfor a particular group of subjects whom have all had chronic kidneydisease or proteinuria, due to, for example, diabetes.

In some embodiments, healthy subjects are selected as the controlsubjects. In some embodiments, controls are age-matched controls.Healthy subject can be used to obtain a reference threshold level ROBO2protein in, for example, a urine or serum sample. A “healthy” subject orsample from a “healthy” subject or individual as used herein is the sameas those commonly understood to one skilled in the art. For example, onemay use methods commonly known to evaluate kidney function, as describedherein, to select control subjects as healthy subjects for diagnosis andtreatment methods described herein. In some embodiments, subjects ingood health with no signs or symptom suggesting chronic kidney diseasecan be recruited as healthy control subjects. The subjects are evaluatedbased on extensive evaluations consisted of medical history, familyhistory, physical and renal examinations by clinicians, laboratorytests. Examples of analyses of chronic kidney disease and/or proteinuriainclude, but are not limited to detecting the level of specific proteinsor molecules in a urine, blood, or serum sample, such as, for example,albumin, calcium, cholesterol, complete blood count (CBC), electrolytes,magnesium, phosphorous, potassium, sodium, or any combination thereof;assays to detect, for example, creatinine clearance; creatinine levels;BUN (blood urea nitrogen); through the use of specific techniques orprocedures, such as an abdominal CT scan, abdominal MRI, abdominalultrasound, kidney biopsy, kidney scan, kidney ultrasound; via detectionof changes in results of assays or tests for erythropoietin, PTH; bonedensity test, or Vitamin D; or any combination of such detection methodsand assays.

Age-matched populations (from which reference values can be obtained)are ideally the same chronological age as the subject or individualbeing tested, but approximately age-matched populations are alsoacceptable. Approximately age-matched populations may be within 1, 2, 3,4, or 5 years of the chronological age of the individual tested, or canbe groups of different chronological ages which encompass thechronological age of the individual being tested.

A subject that is compared to its “chronological age matched group” isgenerally referring to comparing the subject with a chronologicalage-matched within a range of 5 to 20 years. Approximately age-matchedpopulations can be in 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15, or 20 yearincrements (e.g. a “5 year increment” group can serve as the source forreference values for a 62 year old subject might include 58-62 year oldindividuals, 59-63 year old individuals, 60-64 year old individuals,61-65 year old individuals, or 62-66 year old individuals). In a broaderdefinition, where there are larger gaps between different chronologicalage groups, for example, when there are few different chronological agegroups available for reference values, and the gaps between differentchronological age groups exceed the 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15, or20 year increments described herein, then the “chronological age matchedgroup” can refer to the age group that is in closer match to thechronological age of the subject (e.g. when references values availablefor an older age group (e.g., 80-90 years) and a younger age group(e.g., 20-30 years), a chronological age matched group for a 51 year oldcan use the younger age group (20-30 years), which is closer to thechronological age of the test subject, as the reference level.

Other factors to be considered while selecting control subjects include,but not limited to, species, gender, ethnicity, and so on. Hence, insome embodiments, a reference level can be a predetermined referencelevel, such as an average or median of levels obtained from a populationof healthy control subjects that are gender-matched with the gender ofthe tested subject. In some embodiments, a reference level can be apredetermined reference level, such as an average or median of levelsobtained from a population of healthy control subjects that areethnicity-matched with the ethnicity of the tested subject (e.g.,Caucasian, Black, Hispanic, Asian, and Asian-Indian, Pakistani, MiddleEastern and Pacific Islander). In other embodiments, both chronologicalage and gender of the population of healthy subjects are matched withthe chronological age and gender of the tested subject, respectively. Inother embodiments, both chronological age and ethnicity of thepopulation of healthy subjects are matched with the chronological ageand ethnicity of the tested subject, respectively. In other embodiments,chronological age, gender, and ethnicity of the population of healthycontrol subjects are all matched with the chronological age, gender, andethnicity of the tested subject, respectively.

The process of comparing a level of ROBO2 protein in a biological samplefrom a subject and a reference threshold level for ROBO2 protein can becarried out in any convenient manner appropriate and known to one ofskill in the art. Generally, values of ROBO2 protein levels determinedusing the assays, methods, and systems described herein can bequantitative values (e.g., quantitative values of concentration, such asmilligrams of ROBO2 protein per liter (e.g., mg/L) of sample, or anabsolute amount). Alternatively, values of ROBO2 protein levels can bequalitative depending on the measurement techniques, and thus the modeof comparing a value from a subject and a reference value can varydepending on the measurement technology employed. For example, thecomparison can be made by inspecting the numerical data, by inspectingrepresentations of the data (e.g., inspecting graphical representationssuch as bar or line graphs), and using standard deviations of, forexample, at least one, or at least two standard deviations. In oneexample, when a qualitative assay is used to measure ROBO2 proteinlevels, the levels can be compared by visually comparing the intensityof the colored reaction product, or by comparing data from densitometricor spectrometric measurements of the colored reaction product (e.g.,comparing numerical data or graphical data, such as bar charts, derivedfrom the measuring device).

As described herein, ROBO2 protein levels can be measured quantitatively(absolute values) or qualitatively (relative values). In someembodiments, quantitative values of ROBO2 protein levels in thebiological samples can indicate a given level (or grade) of risk ofchronic kidney disease or proteinuria.

In some embodiments, the comparison is performed to determine themagnitude of the difference between the values from a subject andreference values (e.g., comparing the “fold” or percentage differencebetween the measured ROBO2 protein levels obtained from a subject andthe reference threshold ROBO2 protein value). A fold difference can bedetermined by measuring the absolute concentration of the ROBO2 proteinlevels, and comparing that to the absolute value to the referencethreshold ROBO2 protein level, or a fold difference can be measured bythe relative difference between a reference value and a sample value,where neither value is a measure of absolute concentration, and/or whereboth values are measured simultaneously. For example, an ELISA measuresthe absolute content or concentration of a protein from which a foldchange is determined in comparison to the absolute concentration of thesame protein in the reference. As another example, an antibody arraymeasures the relative concentration from which a fold change isdetermined. Accordingly, the magnitude of the difference between themeasured value and the reference value that suggests or indicates aparticular diagnosis will depend on the method being practiced.

As will be apparent to those of skill in the art, when replicatemeasurements are taken for measurement of ROBO2 protein levels, themeasured values from subjects can be compared with the referencethreshold ROBO2 protein levels, and take into account the replicatemeasurements. The replicate measurements can be taken into account byusing either the mean or median of the measured values.

In some embodiments, the process of comparing can be manual or it can,preferably, be automated. For example, an assay device (such as aluminometer for measuring chemiluminescent signals) can includecircuitry and software enabling it to compare a value from a subjectwith a reference value for ROBO2 protein. Alternately, a separate device(e.g., a digital computer) can be used to compare the measured ROBO2protein levels from subject(s) and the reference threshold levels forROBO2 protein. Automated devices for comparison can include storedreference values for the ROBO2 protein, or can compare the measuredROBO2 protein levels from subject(s) with reference threshold levels forROBO2 protein that are derived from contemporaneously measured referencesamples

In some embodiments, a subject tested for ROBO2 protein levels isassigned into one of two or more groups (statuses) based on the resultsof the assays, methods, and systems described herein. The diagnosticassays, methods, and systems described herein can be used to classifybetween a number of different states.

Accordingly, in some embodiments, determining whether a subject has ahigh risk of having chronic kidney disease or proteinuria (status:low-risk v. high risk) is performed using the diagnostic assays,methods, and systems described herein. Biomarker amounts or patterns ofROBO2 protein determined as being characteristic of various risk states,e.g., high, medium or low, are identified. The risk of developingchronic kidney disease or proteinuria is determined by measuring ROBO2protein alone or in combination with other known biomarkers, and theneither submitting them to a classification algorithm or comparing themwith a reference amount (e.g., a cut off reference amount as disclosedherein) that is associated with the particular risk level.

In some embodiments, provided herein are diagnostic assays, methods, andsystems for determining the severity or stage or risk of having achronic kidney disease or proteinuria in a subject. Each stage ofchronic kidney disease, for example, has a characteristic amount ofROBO2 protein or relative amounts of ROBO2 protein. The stage of adisease is determined by measuring ROBO2 protein, alone or incombination with other biomarkers, and then either submitting them to aclassification algorithm or comparing them with a reference amountand/or pattern of biomarkers that is associated with the particularstage, e.g., how soon the subject will likely develop chronic kidneydisease or proteinuria. For example, one can classify between likely tohave chronic kidney disease or proteinuria within a year (e.g., a poorprognosis) or a subject likely to have chronic kidney disease orproteinuria in the next 5 years.

Additional embodiments of the diagnostic assays, methods, and systemsrelate to the communication of results or diagnoses or both totechnicians, physicians or patients, for example. In certainembodiments, computers are used to communicate assay results ordiagnoses or both to interested parties, e.g., physicians and theirpatients. In some embodiments, the assays are performed or the assayresults analyzed in a country or jurisdiction which differs from thecountry or jurisdiction to which the results or diagnoses arecommunicated, for example. In some embodiments, a risk of having chronickidney disease or proteinuria based on levels of ROBO2 protein in abiological sample from the subject is communicated to the subject afterthe levels or prognosis are obtained. The prognosis or diagnosis can becommunicated to the subject by the subject's treating physician.Alternatively, the prognosis or diagnosis can be sent to the subject byemail or communicated to the subject by phone. A computer can be used tocommunicate the prognosis or diagnosis by email or phone, or via theinternet using a secure gateway patient log-in service. In certainembodiments, the message containing results of the prognosis ordiagnostic test can be generated and delivered automatically to thesubject using a combination of computer hardware and software which willbe familiar to artisans skilled in telecommunications. In certainembodiments of the assays, methods, and systems described herein, all orsome of the method steps, including the assaying of samples, diagnosingof diseases, and communicating of assay results or diagnoses, can becarried out in diverse (e.g., foreign) jurisdictions.

In some embodiments of the diagnostic assays, methods, and systems ofqualifying or assessing a risk of chronic kidney disease or proteinuriadescribed herein, the assays, methods, or systems further comprisemanaging subject treatment based on the determination of the risk ofhaving a chronic kidney disease or proteinuria. Such management includesthe actions of the physician or clinician subsequent to determining thesubjects risk of having chronic kidney disease or proteinuria. Forexample, if a physician makes a diagnosis of the subject at risk ofchronic kidney disease or proteinuria, then a certain regimen oftreatment can follow. A suitable regimen of treatment can include,without limitation, a supervised exercise program; control of bloodpressure, sugar intake, and/or lipid levels; and drug therapies. In someembodiments, a diagnosis of a risk of having chronic kidney disease orproteinuria can be followed by further testing to determine whether apatient is suffering from a chronic kidney disease, or whether thepatient is suffering from a related disease. Also, if the diagnostictest gives an inconclusive result on the risk of a major adverse eventstatus, further tests may be called for. In some embodiments of thediagnostic assays, methods, and systems of qualifying or assessing arisk of chronic kidney disease or proteinuria described herein, if aphysician makes a diagnosis of the subject not being at risk of chronickidney disease or proteinuria, then no treatment is provided.

The assay and ROBO2 detection methods described herein can be automatedusing robotics and computer directed systems. A biological sample, suchas a urine, plasma, or blood sample, can be injected into a system, suchas a microfluidic device entirely run by a robotic station from sampleinput to output of the result.

Accordingly, also provided herein, in some aspects are systems (andcomputer readable medium for causing computer systems) to perform amethod for determining whether an individual has a chronic kidneydisease or proteinuria or a pre-disposition for a chronic kidney diseaseor proteinuria based on expression profiles or sequence information.

In some aspects, provided herein are systems for assessing if a subjecthas or is at increased risk for chronic kidney disease or proteinuria,the systems comprising: (a) a determination module configured to receivea at least one biological sample and perform at least one analysis onsaid biological sample to measure a level of ROBO2 in the biologicalsample or determine the expression ratio of ROBO2 relative to apre-determined or threshold reference level and to output said measuredlevel or expression ratio; (b) a storage device configured to store dataoutput information from the determination module; (c) a comparisonmodule adapted to receive input from the storage device and compare thedata stored on the storage device with at least one reference thresholdROBO2 level, wherein if the measured ROBO2 protein level is at least thesame or higher than the reference threshold level, the comparison moduleprovides information to an output module that the biological sample isassociated with a subject that deviates from the reference thresholdbiomarker level; and (d) an output module for displaying the informationto the user.

In all aspects of the invention, methods to determine the levels ofROBO2 protein can be performed using an automated machine or system.Such machines and systems generate a report, such as displaying a reporton a visible screen or a printable report which indicates the levels ofROBO2 protein and/or report an increase or the same as a referencethreshold level for ROBO2 protein, and/or if the subject from which thesample was obtained is at risk of chronic kidney disease or proteinuria.

Accordingly, some embodiments described herein also provide for amachine, computer systems and computer readable media for performing thesteps of (i) determining the levels of ROBO2 protein, and (ii)indicating or reporting whether a subject is at risk of having chronickidney disease or proteinuria.

Embodiments of these aspects are described through functional modules,which are defined by computer executable instructions recorded oncomputer readable media and which cause a computer to perform methodsteps when executed. The modules have been segregated by function forthe sake of clarity. However, it should be understood that the modulesneed not correspond to discreet blocks of code and the describedfunctions can be carried out by the execution of various code portionsstored on various media and executed at various times. Furthermore, itshould be appreciated that the modules can perform other functions, thusthe modules are not limited to having any particular functions or set offunctions.

The computer readable media can be any available tangible media that canbe accessed by a computer. Computer readable media includes volatile andnonvolatile, removable and non-removable tangible media implemented inany method or technology for storage of information such as computerreadable instructions, data structures, program modules or other data.Computer readable media includes, but is not limited to, RAM (randomaccess memory), ROM (read only memory), EPROM (erasable programmableread only memory), EEPROM (electrically erasable programmable read onlymemory), flash memory or other memory technology, CD-ROM (compact discread only memory), DVDs (digital versatile disks) or other opticalstorage media, magnetic cassettes, magnetic tape, magnetic disk storageor other magnetic storage media, other types of volatile andnon-volatile memory, and any other tangible medium which can be used tostore the desired information and which can accessed by a computerincluding and any suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable media,or computer readable medium, can define instructions, for example, aspart of one or more programs, that, as a result of being executed by acomputer, instruct the computer to perform one or more of the functionsdescribed herein (e.g., in relation to a system, or computer readablemedium), and/or various embodiments, variations and combinationsthereof. Such instructions can be written in any of a plurality ofprogramming languages, for example, Java, J#, Visual Basic, C, C#, C++,Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like,or any of a variety of combinations thereof. The computer-readable mediaon which such instructions are embodied can reside on one or more of thecomponents of either of the system, or computer readable mediumdescribed herein, can be distributed across one or more of suchcomponents, and can be in transition there between.

The computer-readable media can be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer readable media, or the computer-readable medium, describedabove, are not limited to instructions embodied as part of anapplication program running on a host computer. Rather, the instructionscan be embodied as any type of computer code (e.g., software ormicrocode) that can be employed to program a computer to implementaspects of the present invention. The computer executable instructionscan be written in a suitable computer language or combination of severallanguages. Basic computational biology methods are known to those ofordinary skill in the art and are described in, for example, Setubal andMeidanis et al., Introduction to Computational Biology Methods (PWSPublishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998);Rashidi and Buehler, Bioinformatics Basics: Application in BiologicalScience and Medicine (CRC Press, London, 2000) and Ouelette and BzevanisBioinformatics: A Practical Guide for Analysis of Gene and Proteins(Wiley & Sons, Inc., 2^(nd) ed., 2001).

The functional modules of certain embodiments of the aspects describedherein include a determination module, a storage device, a comparisonmodule and a display module. The functional modules can be executed onone, or multiple, computers, or by using one, or multiple, computernetworks or computer systems.

In some aspects, provided herein are computer systems that can be usedto determine if a subject is likely to have or be at risk of chronickidney disease or proteinuria. In some embodiments, a computer system isconnected to a determination module and is configured to obtain outputdata from a determination module regarding a biological sample, wherethe determination module is configured to detect the levels of ROBO2protein in a biological sample obtained from the subject; and where thecomputer system comprises (a) a storage device configured to store dataoutput from the determination module as well as reference data; wherethe storage device is connected to (b) a comparison module which in someembodiments, is adapted to compare the output data stored on the storagedevice with stored reference data, and in alternative embodiments,adapted to compare the output data with itself, where the comparisonmodule produces report data and is connected to (c) a display module fordisplaying a page of retrieved content (i.e. report data from thecomparison module) for the user on a client computer, wherein theretrieved content can indicate the levels of ROBO2, and/or likelihood ofthe subject experiencing chronic kidney disease or proteinuria in thefuture.

In some embodiments, the determination module has computer executableinstructions to provide expression data, sequence information,information related to sequence information in computer readable form.As used herein, “sequence information” refers to any nucleotide and/oramino acid sequence, including but not limited to full-length nucleotideand/or amino acid sequences, partial nucleotide and/or amino acidsequences, or mutated sequences. Moreover, information “related to” thesequence information includes detection of the presence or absence of asequence (e.g., detection of a mutation or deletion), determination ofthe concentration of a sequence in the sample (e.g., amino acid sequenceexpression levels, or nucleotide (RNA or DNA) expression levels), andthe like. The term “sequence information” is intended to include thepresence or absence of post-translational modifications (e.g.phosphorylation, glycosylation, summylation, farnesylation, and thelike).

As an example, determination modules for determining ROBO2 sequence ornucleic acid expression information can include known systems forautomated sequence analysis including but not limited to Hitachi FMBIO®and Hitachi FMBIO® II Fluorescent Scanners (available from HitachiGenetic Systems, Alameda, Calif.); SPECTRUMEDIX® SCE 9610 FullyAutomated 96-Capillary Electrophoresis Genetic Analysis Systems(available from SpectruMedix LLC, State College, Pa.); ABI PRISM® 377DNA Sequencer, ABI® 373 DNA Sequencer, ABI PRISM® 310 Genetic Analyzer,ABI PRISM® 3100 Genetic Analyzer, and ABI PRISM® 3700 DNA Analyzer(available from Applied Biosystems, Foster City, Calif.); MolecularDynamics FLUORIMAGER™ 575, SI Fluorescent Scanners, and MolecularDynamics FLUORIMAGER™ 595 Fluorescent Scanners (available from AmershamBiosciences UK Limited, Little Chalfont, Buckinghamshire, England);GENOMYXSC™ DNA Sequencing System (available from Genomyx Corporation(Foster City, Calif.); and PHARMACIA ALF™ DNA Sequencer and PharmaciaALFEXPRESS™ (available from Amersham Biosciences UK Limited, LittleChalfont, Buckinghamshire, England).

In some embodiments for determining sequence or protein information,determination modules include systems for protein and DNA analysis. Forexample, mass spectrometry systems including Matrix Assisted LaserDesorption Ionization—Time of Flight (MALDI-TOF) systems; SELDI-TOF-MSProteinChip array profiling systems, e.g. Machines with CIPHERGENPROTEIN BIOLOGY SYSTEM II™ software; systems for analyzing geneexpression data (see for example U.S. 2003/0194711); systems for arraybased expression analysis, for example HT array systems and cartridgearray systems available from Affymetrix (Santa Clara, Calif. 95051)AutoLoader, COMPLETE GENECHIP® Instrument System, Fluidics Station 450,Hybridization Oven 645, QC Toolbox Software Kit, Scanner 3000 7G,Scanner 3000 7G plus Targeted Genotyping System, Scanner 3000 7GWhole-Genome Association System, GENETITAN™ Instrument, GeneChip® ArrayStation, HT Array; an automated ELISA system (e.g. DSX® or DS2® formDynax, Chantilly, Va. or the ENEASYSTEM III®, TRITURUS®, THE MAGO®Plus); Densitometers (e.g. X-Rite-508-Spectro Densitometer®, The HYRYS™2 densitometer); automated Fluorescence in situ hybridization systems(see for example, U.S. Pat. No. 6,136,540); 2D gel imaging systemscoupled with 2-D imaging software; microplate readers; Fluorescenceactivated cell sorters (FACS) (e.g. Flow Cytometer FACSVantage SE,Becton Dickinson); radio isotope analyzers (e.g. scintillationcounters), or a combination thereof.

In some embodiments of this aspect and all other aspects of the presentinvention a variety of software programs and formats can be used tostore the biomarker protein level information on the storage device. Anynumber of data processor structuring formats (e.g., text file ordatabase) can be employed to obtain or create a medium having recordedthereon the sequence information or expression level information.

The ROBO2 expression information or information related to ROBO2expression information determined in the determination module can beread by the storage device. As used herein the “storage device” isintended to include any suitable computing or processing apparatus orother device configured or adapted for storing data or information.Examples of electronic apparatus suitable for use with the presentinvention include stand-alone computing apparatus, datatelecommunications networks, including local area networks (LAN), widearea networks (WAN), cloud storage systems, Internet, Intranet, andExtranet, and local and distributed computer processing systems. Storagedevices also include, but are not limited to: magnetic storage media,such as floppy discs, hard disc storage media, magnetic tape, opticalstorage media such as CD-ROM, DVD, electronic storage media such as RAM,ROM, EPROM, EEPROM and the like, cloud storage systems, general harddisks and hybrids of these categories such as magnetic/optical storagemedia. The storage device is adapted or configured for having recordedthereon sequence information or expression level information. Suchinformation may be provided in digital form that can be transmitted andread electronically, e.g., via the Internet, via a cloud system, ondiskette, via USB (universal serial bus), or via any other suitable modeof communication.

As used herein, “expression level information” refers to any nucleotideand/or amino acid expression level information, including but notlimited to full-length nucleotide and/or amino acid sequences, partialnucleotide and/or amino acid sequences, or mutated sequences. Moreover,information “related to” the expression level information includesdetection of the presence or absence of a sequence (e.g., presence orabsence of an amino acid sequence, nucleotide sequence, or posttranslational modification), determination of the concentration of asequence in the sample (e.g., amino acid sequence levels, or nucleotide(RNA or DNA) expression levels, or level of post translationalmodification), and the like.

As used herein, “stored” refers to a process for encoding information onthe storage device. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising the sequence information or expressionlevel information.

A variety of software programs and formats can be used to store thesequence information or expression level information on the storagedevice. Any number of data processor structuring formats (e.g., textfile or database) can be employed to obtain or create a medium havingrecorded thereon the sequence information or expression levelinformation.

By providing sequence information or expression level information incomputer-readable form, one can use the sequence information orexpression level information in readable form in the comparison moduleto compare a specific sequence or expression profile with the referencedata within the storage device. For example, search programs can be usedto identify fragments or regions of the sequences that match aparticular sequence (reference data, e.g., sequence information obtainedfrom a control sample) or direct comparison of the determined expressionlevel can be compared to the reference data expression level (e.g.,expression level information obtained from a control sample). Thecomparison made in computer-readable form provides a computer readablecomparison result which can be processed by a variety of means. Contentbased on the comparison result can be retrieved from the comparisonmodule to indicate a specific disease or disorder, such as chronickidney disease or proteinuria.

In some embodiments, the reference data stored in the storage device tobe read by the comparison module is ROBO2 sequence or expressioninformation data obtained from a control biological sample of the sametype as the biological sample to be tested. Alternatively, the referencedata are a database, e.g., a part of the entire genome sequence of anorganism, or a protein family of sequences, or an expression levelprofile (RNA, protein or peptide). In some embodiments, the referencedata are sequence information or expression level profiles that areindicative of a specific disease or disorder, such as chronic kidneydisease or proteinuria.

In some embodiments, the reference data are electronically or digitallyrecorded and annotated from databases including, but not limited toGenBank (NCBI) protein and DNA databases such as genome, ESTs, SNPS,Traces, Celara, Ventor Reads, Watson reads, HGTS, and the like; SwissInstitute of Bioinformatics databases, such as ENZYME, PROSITE,SWISS-2DPAGE, Swiss-Prot and TrEMBL databases; the Melanie softwarepackage or the ExPASy WWW server, and the like; the SWISS-MODEL,Swiss-Shop and other network-based computational tools; theComprehensive Microbial Resource database (available from The Instituteof Genomic Research). The resulting information can be stored in arelational data base that may be employed to determine homologiesbetween the reference data or genes or proteins within and amonggenomes.

By providing the levels of ROBO2 in readable form in the comparisonmodule, it can be used to compare with the reference threshold levels ofROBO2 within the storage device. The comparison made incomputer-readable form provides computer readable content which can beprocessed by a variety of means.

The “comparison module” can use a variety of available software programsand formats for the comparison operative to compare ROBO2 sequence orexpression level information determined in the determination module toreference ROBO2 sequence or expression level data. In some embodiments,the comparison module is configured to use pattern recognitiontechniques to compare ROBO2 sequence or expression level data from oneor more entries to one or more reference data patterns. The comparisonmodule can be configured using existing commercially-available orfreely-available software for comparing patterns, and can be optimizedfor particular data comparisons that are conducted. The comparisonmodule provides computer readable information related to the sequenceinformation that can include, for example, detection of the presence orabsence of a sequence (e.g., detection of a mutation or deletion(protein or DNA), information regarding distinct alleles, detection ofpost-translational modification, or omission or repetition ofsequences); determination of the concentration of a sequence in thesample (e.g., amino acid sequence/protein expression levels, ornucleotide (RNA or DNA) expression levels, or levels ofpost-translational modification), or determination of an expressionprofile.

In some embodiments, the comparison module permits the comparison oflevels of ROBO2 from the output data of the determination module withreference threshold level data for each ROBO2.

In some embodiments, the comparison module performs comparisons withmass-spectometry spectra, for example comparisons of peptide fragmentsequence information can be carried out using spectra processed in MATLBwith script called “Qcealign” (see for example WO2007/022248, hereinincorporated by reference) and “Qpeaks” (Spectrum Square Associates,Ithaca, N.Y.), or Ciphergen Peaks 2.1™ software. The processed spectracan then be aligned using alignment algorithms that align sample data tothe control data using minimum entropy algorithm by taking baselinecorrected data (see for example WIPO Publication WO2007/022248, hereinincorporated by reference). The comparison result can be furtherprocessed by calculating ratios. Protein expression profiles can bediscerned.

Any available comparison software can be used, including but not limitedto, the Ciphergen Express (CE) and Biomarker Patterns Software (BPS)package, Ciphergen Biosystems, Inc., CA, USA. Comparative analysis canbe done with protein chip system software (e.g. The Proteinchip suitefor Bio-Rad Laboratories).

The comparison module, or any other module described herein, can includean operating system (e.g., UNIX) on which runs a relational databasemanagement system, a World Wide Web application, and a World Wide Webserver. World Wide Web application includes the executable codenecessary for generation of database language statements (e.g.,Structured Query Language (SQL) statements). Generally, the executableswill include embedded SQL statements. In addition, the World Wide Webapplication can include a configuration file which contains pointers andaddresses to the various software entities that comprise the server aswell as the various external and internal databases which must beaccessed to service user requests. The Configuration file also directsrequests for server resources to the appropriate hardware—as can benecessary should the server be distributed over two or more separatecomputers. In some embodiments, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in somepreferred embodiments, users can directly access data (via Hypertextlinks for example) residing on Internet databases using a HTML interfaceprovided by Web browsers and Web servers.

In some embodiments, the comparison module compares gene expressionprofiles. For example, detection of gene expression profiles can bedetermined using Affymetrix Microarray Suite software version 5.0 (MAS5.0) (available from Affymetrix, Santa Clara, Calif.) to analyze therelative abundance of a gene or genes on the basis of the intensity ofthe signal from probe sets, and the MAS 5.0 data files can betransferred into a database and analyzed with Microsoft Excel andGeneSpring 6.0 software (available from Agilent Technologies, SantaClara, Calif.). The detection algorithm of MAS 5.0 software can be usedto obtain a comprehensive overview of how many transcripts are detectedin given samples and allows a comparative analysis of 2 or moremicroarray data sets.

In some embodiments, the comparison module compares protein expressionprofiles. Any available comparison software can be used, including butnot limited to, the Ciphergen Express (CE) and Biomarker PatternsSoftware (BPS) package (available from Ciphergen Biosystems, Inc.,Freemont, Calif.). Comparative analysis can be done with protein chipsystem software (e.g., The Proteinchip Suite (available from Bio-RadLaboratories, Hercules, Calif.). Algorithms for identifying expressionprofiles can include the use of optimization algorithms such as the meanvariance algorithm (e.g. JMP Genomics algorithm available from JMPSoftware Cary, N.C.).

The comparison module provides computer readable comparison result thatcan be processed in computer readable form by predefined criteria, orcriteria defined by a user, to provide a content based in part on thecomparison result that can be stored and output as requested by a userusing a display module. The display module enables display of a contentbased in part on the comparison result for the user, wherein the contentis a signal indicative of a chronic kidney disease or proteinuria. Suchsignal, can be for example, a display of content indicative of thepresence or absence of a chronic kidney disease or proteinuria on acomputer monitor, a printed page of content indicating the presence orabsence of a chronic kidney disease or proteinuria from a printer, or alight or sound indicative of the presence or absence of a chronic kidneydisease or proteinuria.

The content based on the comparison result can include an expressionprofile of one or more proteins, or an expression profile of one or moregenes. In some embodiments, the content based on the comparison resultis merely a signal indicative of the presence or absence of a chronickidney disease or proteinuria based on ROBO2 protein levels.

In some embodiments, the content based on the comparison result isdisplayed a on a computer monitor. In one embodiment of the invention,the content based on the comparison result is displayed throughprintable media. In one embodiment of the invention, the content basedon the comparison result is displayed as an indicator light or sound.The display module can be any suitable device configured to receive froma computer and display computer readable information to a user.Non-limiting examples include, for example, general-purpose computerssuch as those based on Intel PENTIUM-type processor, Apple computer andtablet devices, Motorola PowerPC, Sun UltraSPARC, Hewlett-PackardPA-RISC processors, any of a variety of processors available fromAdvanced Micro Devices (AMD) of Sunnyvale, Calif., or any other type ofprocessor, visual display devices such as tablet devices, smartphonemobile devices, flat panel displays, cathode ray tubes and the like, aswell as computer printers of various types.

In some embodiments, a World Wide Web browser is used for providing auser interface for display of the content based on the comparisonresult. It should be understood that other modules of the invention canbe adapted to have a web browser interface. Through the Web browser, auser may construct requests for retrieving data from the comparisonmodule. Thus, the user will typically point and click to user interfaceelements such as buttons, pull down menus, scroll bars and the likeconventionally employed in graphical user interfaces. The requests soformulated with the user's Web browser are transmitted to a Webapplication which formats them to produce a query that can be employedto extract the pertinent information related to the sequenceinformation, e.g., display of an indication of the presence or absenceof mutation or deletion (DNA or protein); display of expression levelsof an amino acid sequence (protein); display of nucleotide (RNA or DNA)expression levels; display of expression, SNP, or mutation profiles, orhaplotypes, or display of information based thereon. In one embodiment,the sequence information of the reference sample data is also displayed.

In some embodiments, the display module displays the comparison resultand whether the comparison result is indicative of a disease, e.g.,whether the expression profile of ROBO2 is indicative of chronic kidneydisease or proteinuria.

In some embodiments, the content based on the comparison result that isdisplayed is a signal (e.g. positive or negative signal) indicative ofthe presence or absence of a chronic kidney disease or proteinuria, thusonly a positive or negative indication can be displayed.

Thus, provided herein are systems (and computer readable medium forcausing computer systems) to perform assays and methods for determiningwhether an individual has a chronic kidney disease or proteinuria or apre-disposition, for a chronic kidney disease or proteinuria based onexpression profiles or sequence information.

Systems and computer readable medium, are merely an illustrativeembodiments of the invention for performing assays and methods ofdetermining whether an individual has a specific disease or disorder ora pre-disposition, for a specific disease or disorder based onexpression profiles or sequence information, and is not intended tolimit the scope of the invention. Variations of systems, and computerreadable medium, are possible and are intended to fall within the scopeof the invention.

The modules of the system or used in the computer readable medium, canassume numerous configurations. For example, function can be provided ona single machine or distributed over multiple machines.

Robo2 is a Podocyte Protein Localized to the Basal Cell Surface of MousePodocyte

During kidney development, Robo2 mRNA is expressed in the metanephricmesenchyme surrounding the arborizing ureteric bud and later in theproximal end of the S-shaped body (Piper et al., 2000), the location ofprimordial podocytes. To investigate whether Robo2 is also involved inpodocyte maturation, in addition to its role in early kidney induction,we performed in situ hybridization and found Robo2 mRNA was expressed inthe capillary loop stage of developing glomeruli of mouse embryos atembryonic day 16.5 (E16.5) (FIGS. 5A and 5B). Robo2 protein becamedetectable by immunofluorescence staining in the developing glomerulusaround E14.5 and reached peak expression at E16.5 (FIGS. 5C-5E).Although the expression decreased after E17.5 during development (FIG.5F), specific Robo2 expression was maintained in glomeruli after birthand was detectable in adult mice at 5 weeks of age (FIGS. 5G, 5H,5L-5M).

To determine the cellular localization of Robo2 in the developingglomerulus, we performed dual-label immunohistochemistry with glomerularcell type specific markers. We found that Robo2 protein was co-localizedwith nephrin (FIGS. 1A-1C) and podocin (FIGS. 1D-1F), two podocyteslit-diaphragm associated proteins. Robo2 was also co-expressed in theglomeruli with the nephrin-interacting adaptor protein Nck (FIGS. 1G,1I) and with WT1, a constituent of podocyte nuclei (FIGS. 5H-5K).Dual-labeling with antibodies against nidogen, a basement membranemarker (FIGS. 1J-1L and 1P) and Pecam1, an endothelial cell marker(FIGS. 1M-1O, 5M) showed that Robo2 was localized adjacent to theexternal surface of the glomerular basement membrane and absent fromendothelial cells. High-resolution confocal microscopy furtherdemonstrated that subcellular Robo2 was most abundant on the basalsurface of podocytes (FIG. 1Q). Immunogold electron microscopy ofpostnatal mouse kidneys with an antibody against the cytoplasmic domainestablished that Robo2 was localized to podocyte foot processes close tothe cytoplasmic face of the slit-diaphragms (FIG. 1R). These resultsdemonstrate that Robo2 is a podocyte protein and its basal subcellularlocalization in the foot processes indicates that it plays a role inregulating podocyte foot process structure.

Robo2 Intracellular Domain Interacts Directly with SH3 Domains ofAdaptor Protein Nck

Nephrin extracellular domain engagement leads to tyrosinephosphorylation of its intracellular domain by Src kinases andrecruitment of the SH2 domain of the adaptor protein Nck, which in turninduces actin polymerization (Jones et al., 2006; Verma et al., 2006).Nck bears one SH2 domain in the C-terminus and three SH3 domains nearthe N-terminus. Actin polymerization is mediated by the SH3 domains ofNck (Rivera et al., 2004), which can recruit various cytoskeletonregulators including N-WASP and Pak (Jones et al., 2006). Previousstudies have shown that the SH3 domains of the Drosophila Nck homologDreadlock (Dock) also directly interact with the intracellular domain ofRobo to inhibit actin polymerization (Fan et al., 2003; Yang and Bashaw,2006).

We tested whether mammalian Nck can also interact directly with Robo2 inthe podocyte to regulate the F-actin cytoskeleton. To answer thisquestion, we used a yeast two-hybrid assay to examine if Robo2interacted with Nck. Since two mammalian Ncks (i.e. Nck1, Nck2) sharesimilar structure and function in kidney development (Jones et al.,2006), we used Nck1 in this study and observed that the intracellulardomain of Robo2 directly interacted with Nck1 (FIGS. 2A-2C). Bindingsite mapping in Robo2 for Nck1 showed that the sequence from amino acid1085 to 1301, which contains 4 proline-rich motifs, was crucial for theinteraction (FIGS. 2A and 2C). Absence of this proline-rich regionprevented its interaction with Nck1 (FIG. 2A). Binding site mapping inNck1 for Robo2 showed that the first two SH3 domains were required forits interaction with Robo2 because deleting either or both of themabrogated the interaction (FIG. 2B). Thus Robo2 and Nck1 interaction wasmediated by two well-characterized protein domains, the SH3 domains andproline-rich motifs (FIG. 2C). CD2AP, another podocyte adaptor protein,also bears three SH3 domains in its N-terminus (Shih et al., 2001), butwe did not detect any interaction between CD2AP and Robo2 in either theyeast two-hybrid or co-precipitation assays. These observations indicatethat the binding between Robo2 and Nck1 in the podocyte is a specificinteraction.

Full-Length Robo2 Forms a Complex with Nephrin Through Nck

We confirmed the interaction between Robo2 and Nck by pull down andco-precipitation assays. His- and myc-tagged human full-length Robo2(His-myc-Robo2) or his- and myc-tagged human Robo2 with a deletion ofthe Nck1 binding domain (His-myc-Robo2-ΔNBD) were expressed in HEKcells. Transfected HEK cells were stimulated with Slit2 conditionedmedium (prepared from Slit2 stably transfected cells) to activate Robo2and increase Nck binding (Fan et al., 2003). Nck was pulled down withHis-mycRobo2 from the HEK cell lysates using Ni-NTA beads but not withHis-myc-Robo2-ΔNBD (FIG. 2D). Since the SH2 domain of Nck interacts withphosphotyrosines in the nephrin cytoplasmic domain (NCD) (Jones et al.,2006; Verma et al., 2006), we examined whether Robo2 formed a complexwith nephrin through Nck using a co-precipitation assay. To establishproof of principle, we co-expressed Robo2 and nephrin in HEK cells withFyn kinase to increase nephrin phosphorylation (Verma et al., 2006).Pull-down of His-myc-Robo2 from the HEK cell lysates with Ni-NTA beadsco-precipitated Nck and nephrin when Fyn was expressed (FIG. 2E). In thereverse order, pulling down His-myc-nephrin co-precipitated Nck andRobo2 when Fyn kinase was expressed (FIG. 2F). Furthermore, theprecipitates prepared with the anti-Nck antibody contained both Robo2and nephrin when Fyn was over-expressed (FIG. 6A). These data indicatethat nephrin, Nck, and Robo2 form a complex in vitro. To validate thesefindings in vivo, we immunoprecipitated Robo2 from newborn mouse kidneylysates and found that Nck and nephrin were co-precipitated (FIG. 2G).Conversely, the precipitates prepared with the anti-nephrin antibodyalso contained Nck and Robo2 (FIG. 2H). Since nephrin is uniquelyexpressed in podocytes, and Nck and Robo2 are also localized in thesecells in the kidney, these results indicate that nephrin, Nck, and Robo2are able to form a complex in podocytes.

To determine the role of Slit2 in the formation of the Robo2-Nck-nephrinprotein complex, His-myc-Robo2, nephrin, and Fyn were co-expressed inHEK cells that were stimulated with Slit2 conditioned medium or controlconditioned medium without Slit2 prior to co-precipitation (FIG. 2I). Weobserved that Slit2 stimulation increased Robo2 binding to Nck andcomplex formation with nephrin. Both ratios of Nck1 versus Robo2 andnephrin versus Robo2 were increased after Slit2 stimulation (FIG. 2J).Consistent with this finding, we observed that Slit2 was stronglyexpressed in newborn mouse glomeruli (FIGS. 6B, 6C).

Slit2-Robo2 Signaling Inhibits Nephrin-Induced Actin Polymerization

Since Slit binds Robo to recruit Dock and srGAPs to inhibit actinpolymerization (Fan et al., 2003), we wished to test whether Robo2 alsorecruits Nck to inhibit actin polymerization in mammalian cells, anopposite role to nephrin that promotes actin polymerization. To addressthis question, we studied actin polymerization by analyzing F-actintails in cells expressing the CD16/7-NCD chimeric protein as previouslydescribed (Jones et al., 2006; Verma et al., 2006). This model utilizesthe extracellular and transmembrane domains of the human immunoglobulinFc receptors CD 16 and CD7 fused to the nephrin cytoplasmic domain(NCD). CD 16/7-HA, in which NCD was replaced by an HA tag, was used as anegative control. These chimeric proteins were co-expressed with Robo2in HEK cells and clustered by treatment with anti-CD16 antibody and asecondary antibody conjugated to rhodamine. We first examined ifclustering of the nephrin cytoplasmic domain could recruit Robo2. Weobserved that engagement of CD 16/7-NCD brought Robo2 into the clusterssince most of the Robo2 co-localized with the CD16/7-NCD clusters (FIGS.6D-6H). However, no colocalization of the Robo2 was observed either withthe CD16/7-HA control (FIG. 6E) or with the Robo2-ANBD construct (FIG.6G), in which the Robo2 Nck binding domain (NBD) was deleted.Interestingly, in the absence of Slit2, colocalization of CD16/7-NCD andRobo2 was significantly reduced (FIG. 6I). These data provide furtherevidence that the nephrin cytoplasmic domain is able to complex with theRobo2 intracellular domain in the presence of Slit2 and validates themodel to determine if the formation of a Robo2-Nck-nephrin complexaffects actin polymerization.

HEK cells expressing CD16/7-NCD and Robo2 were stimulated with Slit2 orcontrol conditioned medium without Slit2 while clustered by theanti-CD16 antibody. Actin polymerization was evaluated by quantifyingthe number of HEK cells with visible F-actin tails (Rivera et al.,2004). We observed that ˜80% of the CD16/7-NCD clustered cells formedF-actin tails that could be revealed by phalloidin staining aspreviously reported (Jones et al., 2006; Verma et al., 2006). Upon Slit2stimulation, however, the number of cells with F-actin tails wassignificantly reduced to approximately 40% (FIGS. 3A and 3C). Only a fewcells were observed to contain shorter F-actin tails when the controlCD16/7-HA proteins were clustered (FIGS. 3B and 3C). To furtherinvestigate whether this inhibition of actin polymerization requiredNck, we repeated this assay using Robo2 without Nck binding domain(Robo2-ΔNBD) to determine if blocking of Nck binding to Robo2 wouldprevent Slit2-Robo2 inhibition on nephrin-induced actin polymerization.CD16/7-NCD was co-expressed with either full-length Robo2 (FIG. 7A) orRobo2-ΔNBD (FIG. 7B) in HEK cells. We observed that deletion of Nckbinding domain in Robo2 significantly compromised Slit2-Robo2 inhibitionon nephrin-induced actin polymerization (FIG. 7C).

Previous study has shown that nephrin is linked to the F-actincytoskeleton (Yuan et al., 2002). To determine if Slit2-Robo2 signalingcould inhibit F-actin associated to nephrin, we immunoprecipitatedCD16/7-NCD and CD16/7-HA with anti-CD16 antibody and examined the amountof F-actin in the precipitates by Western blot. We observed that theabundance of F-actin associated with nephrin was significantly reducedupon Slit2 stimulation (FIGS. 3D and 3E). Conversely, in vivoimmunoprecipitation assay showed that F-actin associated with nephrinimmunoprecipitated by an anti-nephrin antibody from Robo2 newborn nullmouse kidneys was significantly increased compared with that from wildtype or Robo2 heterozygous mouse kidneys (FIGS. 3F and 3G). Takentogether, these results indicate that Slit2-Robo2 signaling inhibitsnephrin-induced actin polymerization.

Loss of Robo2 in Podocytes Causes Altered Foot Process Structure in Mice

We and others have previously shown that almost all Robo2 homozygousnull mice in mixed genetic background die shortly after birth due to asevere CAKUT phenotype (Grieshammer et al., 2004; Lu et al., 2007; Wanget al., 2011). After breeding mice with a Robo2^(del5) mutant allele forfive generations onto C57BL/6 genetic background, mating ofRobo2^(del5/+) heterozygous parents revealed three Robo2^(del5/del5)homozygous null mice that survived to 3 weeks (among a total of 160 miceanalyzed at weaning). To determine if Robo2 was required for podocytefoot process formation during development, we examined theultrastructure of glomeruli in Robo2 null mice at birth and 3 weeks ofage. Although the podocyte body, foot processes and slit-diaphragm wereformed at birth, transmission electron microscopy showed focal footprocess effacement in newborn Robo2^(del5/del5) homozygous null mice(FIGS. 8A-8F). By scanning electron microscopy, we observed irregularinterdigitating foot processes in Robo2^(del5/del5) homozygous null miceat birth and 3 weeks of age (FIGS. 4A-4H). These findings indicate thatRobo2 is required for normal podocyte foot process patterning duringkidney development.

To examine the role of Robo2 in the maintenance of foot processstructure in mature glomeruli, we generated podocyte specific Robo2knockout mice by crossing conditional Robo2^(flox/flox) mice withRobo2^(del5/+); Tg^(Nphs2-Cre/+) heterozygous mice carrying apodocin-Cre transgene. Twenty podocyte specific Robo2 mutant mice withRobo2^(del5/flox); Tg^(Nphs2-Cre/+) genotype and 20 littermate controlmice were analyzed up to one year of age. Podocyte specific Robo2knockout mice were viable and fertile. However, they displayed unusuallybroad podocyte foot processes and focal segmental foot processeffacement at one month (FIGS. 4I-4M). At 6 weeks of age the mutant micedeveloped significant microalbuminuria, which was detected by both ELISAand Western blot analyses (FIGS. 4N and 4O). In addition, scanningelectron microscopy revealed foot process patterning defects in Robo2podocyte specific knockout mice. Instead of orderly zipper-likeinterdigitating secondary foot processes in the wild-type, Robo2podocyte specific knockout mice displayed irregular and disorganizedfoot process interdigitation patterning at one month (FIGS. 8G-8J).These defects became more obvious over time. At seven months of age,overtly disorganized, shorter, and meandering foot processes wereobserved in Robo2 podocyte specific knockout mice (FIGS. 8K-8N), whichwere similar to the phenotype of three-week old Robo2 null mice.Although Robo2 podocyte specific knockout mice displayed normal podocytenumber, matrix deposition was significantly increased in glomeruli(FIGS. 8O-8T, Tables 1 and 2). These morphological changes indicate thatRobo2 plays a role in regulating and maintain glomerular podocyte footprocess structure.

Loss of Robo2 Alleviates the Podocyte Structural Defect in Nephrin NullMice

Nephrin homozygous mice develop a characteristic phenotype with dilationof the Bowman's space, abnormally broad foot processes, absence ofglomerular slit-diaphragms, and significant proteinuria (Done et al.,2008; Hamano et al., 2002). Since Robo2 formed a complex with nephrin,and Slit2-Robo2 signaling inhibited nephrin-induced actinpolymerization, we wondered if loss of Robo2 would modify the podocytephenotype in nephrin null mice. To test this hypothesis of a possiblegenetic interaction between Robo2 and nephrin, we generated bothgermline Robo2^(−/−); Nphs1^(−/−) and podocyte specificRobo2^(flox/flox); Tg^(Nphs2-Cre/+); Nphs1^(−/−) double Robo2-nephrinknockout mice. Like Nphs1^(−/−) single homozygote, both Robo2^(−/−);Nphs1^(−/−) (4/4, 100%) and Robo2^(flox/flox); Tg^(Nphs2-Cre/+);Nphs1^(−/−) (3/3, 100%) double knockout mice died within 10 hours afterbirth. Histological analysis, however, revealed that the morphology ofglomeruli in the Robo2^(−/−); Nphs1^(−/−) double homozygous miceappeared relatively normal compared with the phenotype in Nphs1^(−/−)single nephrin homozygous mice, which had a dilated Bowman's space(FIGS. 8U-8X). The number of glomeruli with dilated Bowman's space wassignificantly reduced in Robo2^(−/−); Nphs1^(−/−) double homozygous mice(2/55, 3.6%) compared with nephrin single null mice (31/122, 25.4%)(FIG. 8Y and Table 3). In addition, analysis of glomerularultrastructure by scanning electron microscopy showed that theinterdigitating podocyte foot process structure was observed in only 1(6.67%) of 15 glomeruli from nephrin single homozygous mice (FIGS. 4Pand 4Q) compared with 100% in Robo2^(−/−) single homozygotes (FIGS. 4Tand 4U) and wild-type controls (FIGS. 4V and 4W). Remarkably, theinterdigitating pattern of the podocyte foot processes was restored in12 (75%) of 16 glomeruli from Robo2^(−/−); Nphs1^(−/−) double homozygousnewborn mouse kidneys (FIGS. 4R and 4S, Table 4), indicating that aconcomitant loss of Robo2 and nephrin alleviated the podocyte footprocess structural phenotype in these mice. These findings indicate thatthe Robo2-Nck-nephrin physical interactions described above have asubstantial effect on podocyte foot process morphology in vivo when thelevels of expression of Robo2 and nephrin are genetically altered.

Podocytes exhibit a remarkable degree of plasticity. During developmentthey differentiate from simple cuboidal epithelial cells into theelaborate process-bearing cells that we recognize as mature podocytes(Reeves et al., 1978). This plasticity is retained after maturation. Itis seen most graphically as reversible foot process effacement followingexperimental surface charge neutralization with protamine sulfate andrestoration with heparin (Seiler et al., 1975) and during relapse andremission of proteinuria in children with minimal change disease(Nachman et al., 2008). More subtle changes in foot processes probablyoccur under physiological conditions in response to positive andnegative signals in the form of hemodynamic, hormonal or paracrinestimuli. Given the abundance of F-actin in the foot processes, it islikely, without wishing to be bound or limited by theory, that suchstimuli bring about those subtle changes in response to positive andnegative signals transduced to the F-actin cytoskeleton. Too much andunbalanced positive signals may lead to disease phenotype. Indeed,although a physiological ligand has yet to be identified, it is clearthat clustering and phosphorylation of nephrin induces actinpolymerization by recruiting Nck, a mechanism that can be involved inthe proteinuria induced in rats by a nephritogenic monoclonal antibodyto the extracellular domain of nephrin (Topham et al., 1999) and incases of congenital nephrotic syndrome that develop anti-nephrinalloantibodies after renal transplantation (Patrakka et al., 2002).

Our studies described herein reveal another level of negative regulationof podocyte actin polymerization in which Robo2, when bound by Slit,inhibits nephrin-induced actin polymerization. We propose, withoutwishing to be bound or limited by theory, that Slit-Robo2 signaling caninhibit nephrin-induced actin polymerization to maintain normal podocytefoot process structure as follows: Under physiological conditions (e.g.during foot process development), nephrin engagement leads tophosphorylation of the intracellular Y1191/1208/1232 to which the NckSH2 domain binds (Jones et al., 2006; Verma et al., 2006). Nck in turnrecruits cytoskeleton regulators such as N-WASP through its SH3 domainsto promote actin polymerization for podocyte foot-process extension orspreading (FIG. 8Z). Local secretion and binding of Slit increases theinteraction of Robo2 with Nck through its proline rich region and thefirst two SH3 domains of Nck. Sequestering the first two SH3 domains ofNck by Robo2 would inhibit nephrin-Nck mediated actin polymerization anddecrease F-actin associated with nephrin to maintain a dynamic andbalanced F-actin cytoskeleton and normal podocyte foot process structure(FIG. 8Z). In addition to direct inhibition of nephrin induced actinpolymerization through Nck, Slit-Robo2 signaling can inactivate actinpolymerization through other pathways, such as recruiting Ena, Abl,srGAPs to negatively regulate F-actin cytoskeleton as previouslyreported (Bashaw et al., 2000; Wong et al., 2001). In the absence ofSlit2-Robo2 signaling (e.g., when Robo2 is knocked out), the inhibitoryeffects of Robo2 on nephrin induced polymerization is lost. The SH3domains of Nck are able to interact with downstream cytoskeletalregulators to increase actin polymerization (FIG. 8Z), which can explainthe altered podocyte foot process structure identified in Robo2 mutantmice. Our results described herein thus support a mechanism wherebySlit-Robo signaling can regulate podocyte plasticity by negativelyregulating F-actin cytoskeleton, which is similar to the role ofSlit-Robo signaling in axon growth cone pathfinding (Guan and Rao,2003). The pathological finding of increased matrix deposition in theRobo2 mutant mouse glomeruli likely represents a secondary response.

Although it is clear from our studies described herein that Robo2localizes to the basal surface of podocytes and forms a complex withother established foot process slit-diaphragm proteins through itsintracellular domain, it remains uncertain if it actually forms part ofthe slit-diaphragm itself. Interestingly, the extracellular domain ofRobo2 resembles that of nephrin, which has eight immunoglobulin(Ig)-like motifs and one fibronectin domain whereas Robo2 has fiveIg-like motifs and three fibronectin domains (FIG. 8Z) (Tryggvason etal., 2006). We have tested the interaction between the intracellulardomain of Robo2 and the cytoplasmic domain of nephrin in the yeasttwo-hybrid assay. Our biochemical data (FIGS. 2E and 2F) also did notsupport a direct interaction between these two receptors in vitro.However, it is possible that the extracellular domain of Robo2 canassociate with the extracellular domain of nephrin in vivo on the cellmembranes of adjacent foot processes through a trans-interaction in theslit-diaphragm.

We found that Robo2 homozygous null and podocyte specific knockout micedeveloped an altered foot process interdigitating pattern, a phenotypethat is different from that of the nephrin null mice (Hamano et al.,2002; Done, 2008). This is not surprising since nephrin and Robo2 playopposite roles in regulating podocyte F-actin cytoskeleton. Whilenephrin signaling induces localized actin polymerization, Slit2-Robo2signaling acts as a negative regulator on actin polymerization tomaintain podocyte foot process plasticity and dynamics. It is noteworthythat a similar foot process organization defect is observed in mice inwhich the actin-depolymerizing factor Cofilin-1, another negativeregulator of the F-actin cytoskeleton in podocytes, is knocked out (Garget al., 2010). This indicates that the absence of either an actinpolymerization promoting factor such as nephrin signaling or aninhibitory factor such as Robo2 signaling will affect the normalstructure of podocytes. Thus the balance between positive and negativeF-actin cytoskeleton regulators in podocytes is important to maintainnormal foot process structure. Regaining this balance by knocking outboth positive (nephrin) and negative (Robo2) signals can explain therestoration of podocyte foot process interdigitation and milderglomerular phenotype in the Robo2-nephrin double knockout mice. Ourstudies described herein demonstrate the dual roles of nephrin as anessential component of the slit-diaphragm to control glomerularpermselectivity on the one hand (Tryggvason et al., 2006) and as aregulator of foot process morphology through its interaction with theactin cytoskeleton (Jones et al., 2006; Verma et al., 2006) on theother. While Robo2 signaling clearly counters the positive signalingeffects of nephrin on the foot processes, it remains to be determined ifit also influences slit-diaphragm integrity.

Accordingly, as described herein, we have identified Robo2 as a newcomponent of the podocyte intercellular junction in the kidney. We havedemonstrated interactions between Robo2 and nephrin using biochemical,functional, and genetic techniques and have shown that Slit2-Robo2signaling inhibits nephrin-induced actin dynamics. Our results indicatethat Robo2 signaling acts as a negative regulator on nephrin to modulatepodocyte foot process architecture. This study extends our understandingof the role of Slit-Robo signaling and identifies a novel crosstalkmechanism by which guidance cue receptor Robo might influence F-actincytoskeleton dynamics.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. As usedin this specification and the appended claims, the singular forms “a,”“an,” and the include plural references unless the context clearlydictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that could beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Embodiments of the various aspects described herein can be illustratedby the following numbered paragraphs:

-   -   2. A method for the treatment of chronic kidney disease in a        subject in need thereof, comprising administering to a subject        having or at risk for a chronic kidney disease a therapeutically        effective amount of a composition comprising a ROBO2 inhibitor.    -   3. A method for the reduction of proteinuria in a subject in        need thereof, comprising administering to a subject having or at        risk for proteinuria a therapeutically effective amount of a        composition comprising a ROBO2 inhibitor.    -   4. The method of any one of paragraphs for 2, wherein the ROBO2        inhibitor is a blocking antibody or antigen-binding fragment        thereof specific for ROBO2, an antisense molecule specific for        ROBO2, a short interfering RNA (siRNA) specific for ROBO2, a        small molecule inhibitor of ROBO2, a ROBO2 inhibitory        polypeptide, or a ROBO2 structural analog.    -   5. The method of any one of paragraphs 1-3, wherein the ROBO2        inhibitor blocks or reduces binding of ROBO2 to SLIT, to Nck, or        to both.    -   6. The method of any one of paragraphs 1-4, wherein the ROBO2        inhibitor is specific for the Ig1 SLIT binding domain, the Ig1        and Ig2 SLIT binding domains, the Nck intracellular binding        domain, or any combination thereof.    -   7. The method of paragraph 3, wherein the ROBO2 inhibitory        polypeptide is a dominant negative ROBO2 fusion protein, a        polypeptide comprising a ROBO2 extracellular domain without the        intracellular domain, or a polypeptide comprising a ROBO2        intracellular domain without the extracellular domain.    -   8. The method of any one of paragraphs 1-6, wherein the subject        having or at risk for a chronic kidney disease has diabetic        nephropathy or high blood pressure.    -   9. The method of any one of paragraphs 1-7, further comprising        administering to the subject an additional therapeutic agent.    -   10. The method of paragraph 8, wherein the additional        therapeutic agent is an angiotensin-converting enzyme (ACE)        inhibitor or an angiotensin II receptor blocker (ARB).    -   11. A method comprising:        -   a. assaying a biological test sample from a subject to            determine an expression level of ROBO2 polypeptide or an RNA            encoding a ROBO2 polypeptide;        -   b. determining whether the expression level of ROBO2            polypeptide or the expression level of the RNA encoding a            ROBO2 polypeptide in the biological test sample is above a            reference threshold level; and        -   c. diagnosing the subject as in need of treatment or therapy            for chronic kidney disease.    -   12. The method of paragraph 10, wherein assaying the expression        level of ROBO2 polypeptide is performed using an antibody or        antigen-binding fragment thereof specific for the ROBO2        polypeptide.    -   13. The method of paragraph 10, wherein assaying the expression        level of the RNA encoding a ROBO2 polypeptide is performed using        PCR or a hybridization assay.    -   14. The method of any one of paragraphs 10-12, wherein the        biological test sample is a kidney biopsy, urine, blood, serum        sample, or cells pelleted from a urine sample.    -   15. The method of any one of paragraphs 10-13, wherein the        expression level of ROBO2 polypeptide or the expression level of        the RNA encoding a ROBO2 polypeptide is at least 20% above the        reference threshold level.    -   16. The method of any one of paragraphs 10-13, wherein the        expression level of ROBO2 polypeptide or the expression level of        the RNA encoding a ROBO2 polypeptide is at least two standard        deviations above the reference threshold level.    -   17. An assay comprising:        -   a. contacting a biological test sample isolated from a            subject with a reagent that detects ROBO2 polypeptide or an            RNA encoding a ROBO2 polypeptide; and        -   b. measuring the level of ROBO2 polypeptide or an RNA            encoding a ROBO2 polypeptide,        -   c. wherein an increased level of said ROBO2 polypeptide or            said RNA encoding a ROBO2 polypeptide, relative to a normal            biological sample, identifies a subject having chronic            kidney disease and/or progression of chronic kidney disease            or proteinuria.    -   18. The assay of paragraph 16, wherein detecting the expression        level of ROBO2 polypeptide is performed using an antibody or        antigen-binding fragment thereof specific for the ROBO2        polypeptide.    -   19. The assay of paragraph 16, wherein detecting the expression        level of the RNA encoding a ROBO2 polypeptide is performed using        PCR or a hybridization assay.    -   20. The assay of any one of paragraphs 16-18, wherein the        biological test sample is a kidney biopsy, urine, blood, serum        sample, or cells pelleted from a urine sample.    -   21. The assay of any one of paragraphs 16-19, wherein the        expression level of ROBO2 polypeptide or the expression level of        the RNA encoding a ROBO2 polypeptide is at least 20% above the        reference threshold level.    -   22. The assay of any one of paragraphs 16-19, wherein the        expression level of ROBO2 polypeptide or the expression level of        the RNA encoding a ROBO2 polypeptide is at least two standard        deviations above the reference threshold level.    -   23. The assay of any one of paragraphs 16-21, wherein the        subject has been diagnosed with diabetes or high blood pressure.    -   24. A system for determining if a subject is at risk for chronic        kidney disease or proteinuria, or has chronic kidney disease        comprising:        -   a. a measuring module configured to determine the expression            level of ROBO2 polypeptide or the expression level of the            RNA encoding a ROBO2 polypeptide in a biological sample            obtained from a subject;        -   b. a comparison module configured to receive said expression            level of ROBO2 polypeptide or the expression level of the            RNA encoding a ROBO2 polypeptide determined by the measuring            module and perform at least one analysis to determine            whether the expression level of ROBO2 polypeptide or the            expression level of the RNA encoding a ROBO2 polypeptide is            greater than a pre-determined reference level or ratio, and            to provide a retrieved content; and        -   c. a display module for displaying a content based the data            output from said comparison module, wherein the content            comprises a signal indicative that the expression level or            ratio of ROBO2 polypeptide or RNA is greater than the            pre-determined reference level or ratio, or a signal            indicative that the level or expression ratio of ROBO2 is            not greater than the reference level or pre-determined            ratio.    -   25. The system of paragraph 23, wherein the content displayed on        said display module further comprises a signal indicative of the        subject being recommended to receive a particular treatment        regimen.    -   26. A system for determining if a subject is at risk for chronic        kidney disease or proteinuria, or has chronic kidney disease        comprising:        -   a. a determination module configured to receive at least one            test sample obtained from a subject and perform at least one            analysis on said at least one test sample to determine the            presence or absence of either of the following conditions:            -   i. an expression ratio of ROBO2 greater than a                pre-determined ratio, or            -   ii. an expression level of ROBO2 greater than a                pre-determined level        -   b. a storage device configured to store data output from            said determination module; and        -   c. a display module for displaying a content based on the            data output from said determination module, wherein the            content comprises a signal indicative that the expression            ratio of ROBO2 is greater than the pre-determined ratio or            level of ROBO2 greater than a pre-determined level, or a            signal indicative that the expression ratio of ROBO2 is not            greater than the pre-determined ratio or not greater than a            pre-determined level.    -   27. The system of paragraph 25, wherein the content displayed on        said display module further comprises a signal indicative of the        subject being recommended to receive a particular treatment        regimen.    -   28. A method for treating a human subject with a risk of chronic        kidney disease or proteinuria, comprising administering a        treatment or therapy to prevent the occurrence of chronic kidney        disease or proteinuria to a human subject who is determined to        have a level of ROBO2 protein above a reference threshold level.    -   29. The method of paragraph 27, wherein the level of ROBO2        protein is at least 20% above the reference level.    -   30. The method of paragraph 27, wherein the level of ROBO2        protein is at least two standard deviations above the reference        level.    -   31. The method of any one of paragraphs 27-29, wherein the        treatment or therapy to prevent the occurrence of chronic kidney        disease or proteinuria comprises a ROBO2 inhibitor.    -   32. The method of paragraph 30, wherein the ROBO2 inhibitor is a        blocking antibody or antigen-binding fragment thereof specific        for ROBO2, an antisense molecule specific for ROBO2, a short        interfering RNA (siRNA) specific for ROBO2, a small molecule        inhibitor of ROBO2, a ROBO2 inhibitory polypeptide, or a ROBO2        structural analog.    -   33. The method of any one of paragraphs 30-31, wherein the ROBO2        inhibitor blocks or reduces binding of ROBO2 to SLIT, to Nck, or        to both.    -   34. The method of any one of paragraphs 30-32, wherein the ROBO2        inhibitor is specific for the Ig1 SLIT binding domain, the Ig1        and Ig2 SLIT binding domains, the Nck intracellular binding        domain, or any combination thereof.    -   35. The method of paragraph 31, wherein the ROBO2 inhibitory        polypeptide is a dominant negative ROBO2 fusion protein, a        polypeptide comprising a ROBO2 extracellular domain without the        intracellular domain, or a polypeptide comprising a ROBO2        intracellular domain without the extracellular domain.    -   36. A ROBO2 inhibitor for use in treating a chronic kidney        disease.    -   37. A ROBO2 inhibitor for use in treating proteinuria.    -   38. The use of any one of paragraphs 35 or 36, wherein the ROBO2        inhibitor is a blocking antibody or antigen-binding fragment        thereof specific for ROBO2, an antisense molecule specific for        ROBO2, a short interfering RNA (siRNA) specific for ROBO2, a        small molecule inhibitor of ROBO2, a ROBO2 inhibitory        polypeptide, or a ROBO2 structural analog.    -   39. The use of any one of paragraphs 35-37, wherein the ROBO2        inhibitor blocks or reduces binding of ROBO2 to SLIT, to Nck, or        to both.    -   40. The use of any one of paragraphs 35-38, wherein the ROBO2        inhibitor is specific for the Ig1 SLIT binding domain, the Ig1        and Ig2 SLIT binding domains, the Nck intracellular binding        domain, or any combination thereof.    -   41. The use of paragraph 37, wherein the ROBO2 inhibitory        polypeptide is a dominant negative ROBO2 fusion protein, a        polypeptide comprising a ROBO2 extracellular domain without the        intracellular domain, or a polypeptide comprising a ROBO2        intracellular domain without the extracellular domain.    -   42. The use of any one of paragraphs 35-40, wherein the chronic        kidney disease or proteinuria is caused by diabetic nephropathy        or high blood pressure.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Tissue In Situ Hybridization, Immunohistochemistry, andImmunogold Electron Microscopy

In situ hybridization analysis was performed with digoxigenin-labeledRobo2 riboprobes as previously described (Grieshammer et al., 2004).Immunohistochemistry was performed on mouse embryonic kidney tissuesfixed in 4% paraformaldehyde and in adult mouse kidney tissues fixed inmethanol. For immunogold electron microscopy, wild-type mouse kidneyswere dissected and fixed in paraformaldehyde-lysine-periodate (PLP).Ultrathin sections of the mouse kidney were prepared and incubated withgoat anti-Robo2 antibody (DAKO Corporation) and a secondary antibodycoupled to 10 nm colloidal gold (Ted Pella).

Yeast Two-Hybrid, Co-Precipitation, and Actin Polymerization Assays

The DUPLEX-A™ yeast two-hybrid system (OriGene Tech) was used tocharacterize Robo2 and Nck1 interaction according to manufacturer'sinstructions. Cell culture, His-tagged protein co-precipitation, andimmunoprecipitaion were performed as previously reported (Fan et al.,2003). For endogenous immunoprecipitation, mouse newborn kidneys wereutilized. CD16 antibody-mediated crosslinking of CD16/7 fusion proteinsand the actin polymerization assay were performed as previouslydescribed (Jones et al., 2006; Rivera et al., 2004; Verma et al., 2006).

Knockout Mouse Study, Transmission and Scanning Electron Microscopy, andKidney Glomerular Analysis

Mouse protocols were approved by the Institutional Animal Care and UseCommittee (IACUC) at Boston University Medical Center (#14388). Thegeneration and genotyping of Robo2^(flox) conditional allele,Robo2^(del5) (also called Robo2⁻ interchangeably in this paper) germlinemutant allele, and Robo2⁺ wild-type allele were described previously (Luet al., 2007; Wang et al., 2011). To generate Robo2-nephrin doubleknockout mice, Robo2^(+/−) mice were crossed with Nphs1^(+/−) mice thatwere generated previously (Hamano et al., 2002). For transmissionelectron microscopy, kidneys were fixed, incubated in 2% glutaraldehydein 0.15 M sodium cacodylate, dehydrated in graded ethanol, embedded inEpon, sectioned, and stained with uranyl acetate and lead citrate.Ultrathin kidney sections were examined using a JEM-1011 electronmicroscope. For scanning electron microscopy, kidneys were preparedfollowing the standard protocol. For kidney pathological studies,kidneys were fixed in 4% paraformaldehyde, paraffin embedded, sectioned,and stained using standard Periodic acid-Schiff (PAS) or eosinhematoxylin (H&E) methods. For quantification of podocyte number, WT1was used as a podocyte nuclear marker and immunoperoxidase staining wasperformed on kidney sections following the standard protocol. WT1positive podocyte nuclei in each glomerular cross section were counted.For proteinuria analysis, “spot” urine specimens from 6 weeks old micewere examined using a murine albuminuria ELISA quantitation kit(Exocell) according to manufacturer's instruction and urine dipstick(Multistix, Bayer, Ind.) as a screening method.

Tissue In Situ Hybridization and Immunohistochemistry

In situ hybridization analysis was performed with digoxigenin-labeledRobo2 riboprobes as previously described (Grieshammer et al., 2004). TheRobo2 cDNA was linearized with NotI and probes were generated using theDIG DNA labeling and detection kit (Roche Applied Science).Hybridization was performed on 4% paraformaldehyde fixed OCT embeddedmouse embryonic kidney frozen sections. Immunohistochemistry wasperformed on mouse embryonic kidney tissues fixed in 4% paraformaldehydefollowed by 30% sucrose cryoprotection (Mugford et al., 2008) and inadult mouse kidney tissues fixed in methanol. Mouse kidneys embedded inOCT compound were frozen and sectioned using Cryostat at 8-10 μm.Sections were stained with primary antibodies and followed by anappropriate FITC or Cy3 conjugated secondary antibodies. The primaryantibodies used in this study include the ones against ROBO2 (R&DSystem, Abnova, Santa Cruz Biotechnology), nephrin (custom synthesized)(Topham et al., 1999), Nck (Upstate/Millipore), podocin (Sigma), nidogen(Santa Cruz Biotechnology), Pecam1 (BD Biosciences), WT1 (Santa CruzBiotechnology), SLIT2 (Santa Cruz Biotechnology), PDGFR beta (CellSignaling), Synaptopodin (Santa Cruz Biotechnology). Images wereobtained using a Perkin Elmer UltraView LCI multi-point spinning disclaser-scanning confocal microscope and a Zeiss LSM 510 confocal laserscanning microscope with a 60× oil immersion objective.

Immunogold Electron Microscopy

Wild-type mouse kidneys were dissected and fixed inparaformaldehyde-lysine-periodate (PLP) at 4° C. overnight. The tissuewas washed in 1×PBS and dehydrated in graded ethanol and embedded in LRWhite resin (Electron Microscopy Sciences). Ultrathin sections of themouse kidney were prepared and transferred to Formvar-coated gold grids,and blocked with 1% bovine serum albumin and 5% normal goat serum in1×PBS. The sections were then incubated with goat anti-Robo2 antibodywith a 1:50 dilution in DAKO (DAKO Corporation) at 4° C. overnight.Non-immune serum was used as a control. After three washes with 1×PBS,sections were incubated with a IgG secondary antibody coupled to 10 nmcolloidal gold (Ted Pella) for 2 hours at room temperature. Sectionswere finally post-fixed with 1% glutaraldehyde and contrasted withuranyl acetate. Sections were examined with a JEM-1011 transmissionelectron microscope (JEOL, Tokyo, Japan) at 80 kV, and images wereacquired using an AMT digital imaging system (Advanced MicroscopyTechniques, Danvers, Mass.) and imported into Adobe Photoshop.Subcellular localization of Robo2 stained with gold particles inglomeruli was recognized on digital electron micrographs in comparisonwith control micrographs stained with non-immune serum.

Yeast Two-Hybrid Assay

The DUPLEX-A™ yeast two-hybrid system (OriGene Tech, Rockville, Md.) wasused to characterize Robo2 and Nck1 interaction. The cDNAs encoding theintracellular domain of human Robo2 and its truncated forms were clonedinto the pJG4-5 vector at EcoRI/XhoI sites, fusing them to thetranscription activation domain of B42. The cDNAs of human Nck1 and itstruncated forms were cloned into the pEG202 vector at EcoRI/XhoI to fusethem to the DNA binding domain of LexA. The lacZ gene in the constructpSH18-34 and the LEU2 gene in the EGY48 strain yeast genome were used asreporter genes. The pEG202, pSH18-34, and pJG4-5 constructs wereco-transformed into yeast EGY48 cells. The interaction was consideredpositive if the yeast cells turned blue in the presence of X-gal andgrew in the absence of leucine.

Cell Culture, DNA Constructs, Transfection, Co-Precipitation, andWestern Blot Analyses

HEK (293T) cells were transfected at 60% confluency using calciumphosphate transfection. To make C-terminal his- and myc-tagged fusionproteins, full-length human nephrin and Robo2 were cloned into pSecTag Bvector (Invitrogen) at Hind III/EcoR1 and EcoR1/Xho1 restriction sitesrespectively. Robo2-ΔNBD was obtained by deleting the Nck binding domain(FIG. 2C) using QUIKCHANGE site-directed mutagenesis kit (Strategene)according to manufacturer's instructions. Non-tagged Robo2 and Nck1 werecloned into pCS2 vector (Addgene) at EcoR1/Xho1 sites, nephrin atHindIII/EcoR1 sites. Human Fyn and myc-tagged Slit2 constructs have beenreported previously (Li et al., 2008; Wong et al., 2001). CD16/7-NCD andCD16/7-HA constructs were also reported previously (Verma et al., 2006).To detect Robo2 and Nck1 interaction, C-terminal His- and myc-taggedhuman Robo2 or Robo2-ΔNBD was expressed in HEK cells. Forty-eight hourpost-transfection, cells were lysed in the lysis buffer (50 mM NaH₂PO₄,300 mM NaCl, 10 mM Imidazole, 0.5% TX100, 1× protease inhibitor [pH8.0]). Cell lysates were centrifuged for 10 min at 4° C.; supernatantswere incubated with Ni-NTA resin (Qiagen) at 4° C. for 2 hours toprecipitate His-Robo2, NTA resin without Ni was used as a control. Theresin was washed three times with washing buffer (50 mM NaH₂PO₄, 300 mMNaCl, 20 mM Imidazole, 0.5% TX100 [pH 8.0]) and heated at 95° C. for 10min. The precipitates were resolved on SDS-PAGE gels and blotted withrabbit anti-myc, rabbit monoclonal anti-Nck1 (Cell Signaling) antibodiesat a 1:1000 dilution. To examine the triple interaction among Robo2,Nck1, and nephrin, His-myc-Robo2 or His-myc-Robo2-ΔNBD were co-expressedin HEK cells with human nephrin and human Fyn. His-myc-Robo2 wasprecipitated with Ni-NTA beads as described above. To confirm the tripleinteraction, His-myc-nephrin was co-expressed with Robo2, and Fyn in HEKcells and His-myc-nephrin was pulled-down by Ni-NTA beads. Precipitateswere blotted with rabbit polyclonal anti-myc, rabbit monoclonalanti-Nck1, rabbit polyclonal anti-nephrin, mouse monoclonal anti-Robo2(R&D systems), and rabbit polyclonal anti-Fyn (Santa Cruz Biotechnology)antibodies at a 1:1000 dilution. For co-immunoprecipitation ofendogenous proteins, kidneys from newborn mice were homogenized in theRIPA buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5%sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 1× protease inhibitor) onice. Samples were centrifuged for 10 min at 4° C. and the supernatantwas incubated with 1 μg mouse monoclonal anti-Robo2 antibody (R&DSystems) for 1 hour at 4° C. The control goat IgG (Santa CruzBiotechnology) was used as a control. Samples were then mixed with 30 μlof protein A/G Plus agarose bead slurry (Santa Cruz Biotechnology) andfurther incubated for 12 hours at 4° C. Beads were then washed threetimes in the RIPA buffer and proteins were eluted in 1× protein loadingbuffer by heating at 95° C. for 10 min. Precipitates were resolved onSDS-PAGE gels and blotted with mouse anti-Robo2, rabbit anti-nephrin,and rabbit anti-Nck1 antibodies as described above. Actin was blottedwith anti-beta-actin mouse antibody from Sigma. Intensity of the bandswas measured using ImageJ. For proteinuria detection, mice spot urineswere collected and diluted with 1× protein loading buffer at 1:100dilution. Urine proteins were then resolved on SDS-PAGE gels andpurified albumin was used as a control (MP Biomedicals). Gels wereblotted with rabbit anti-albumin polyclonal antibody (MP Biomedicals).

CD16/7-NCD Crosslinking and Actin Polymerization Assay

CD16 antibody-mediated crosslinking of CD16/7 fusion proteins has beendescribed previously (Jones et al., 2006; Rivera et al., 2004; Verma etal., 2006). Briefly, CD16/7-NCD or CD16/7-HA was co-expressed in HEKcells with Robo2. After 24 hours, cells were transferred and seeded onglass coverslips coated with polylysine for another 24 hours. Cells werethen incubated with 1 μg/ml mouse monoclonal anti-CD16 (Beckman Coulter)for 30 min at 37° C., washed once with DMEM, incubated withrhodamine-conjugated secondary antibody (Thermo Scientific) diluted inSlit2 conditioned medium (Wong et al., 2001) or control conditionedmedium for 30 min and fixed in 4% paraformaldehyde in 1×PBS. F-actin wasstained using FITC-conjugated phalloidin (Invitrogen) according tomanufacturer's instruction. The newly formed F-actin bundles stick tothe clustered nephrin (CD16/7-NCD) and look like comet tails (i.e. actintails in the main text) under fluorescence microscope. In thisexperiment, we only analyzed the F-actin bundles formed by clustering ofCD16/7-NCD and attached to the clusters. The cells with F-actin tailswere counted and compared to the total CD16/7-NCD transfected cells. Thequantification formula is: Percentage %=(number of transfected cellswith F-actin tails/total number of cells transfected)×100. Images wereobtained using a LSM510 confocal microscope with a 60× oil immersionobjective.

Generation and Characterization of Robo2 Podocyte Specific Knockout Miceand Robo2-Nephrin Double Knockout Mice

The generation and genotyping of Robo2^(flox) conditional allele,Robo2^(del5) (also called Robo2⁻ interchangeably in this paper) germlinemutant allele, and Robo2⁺ wild-type allele were described previously (Luet al., 2007; Wang et al., 2011). Standard breeding scheme was followedto generate Robo2 podocyte specific Robo2^(del5/flox); Tg^(Nphs2-Cre+)knockout mice, which carry one Robo2^(del5) allele and one Robo2^(flox)allele. In this compound mutant, podocyte specific Cre recombinasedriven by podocin promoter deletes only the Robo2^(flox) allele tofacilitate the penetrance of a phenotype because the other allele,Robo2^(del5), has been deleted ubiquitously from germline expression.The authenticity of Robo2^(del5/flox); Tg^(Nphs2-Cre/+) mice wasdetermined by tail DNA genotyping for the presence of Robo2^(del5) andRobo2^(flox) alleles as well as Tg^(Nphs2-Cre) transgene. F2 littermatesRobo2^(flox/+) mice without Robo2^(del5) allele and Tg^(Nphs2-Cre)transgene were used as controls. To generate Robo2-nephrin doubleknockout mice, Robo2^(+/−) heterozygous mice were crossed withNphs1^(+/−) heterozygous mice that were generated previously (Hamano etal., 2002). After the generation of Robo2^(+/−); Nphs1^(+/−) doubleheterozygous mice, the cross of double heterozygous mice was performedto generate Robo2^(−/−); Nphs1^(−/−) double homozygous mice as well asNphs1^(−/−) single homozygous, Robo2^(−/−) single homozygous, andRobo2^(+/+); Nphs1^(+/+) wild-type controls. Mouse protocols wereapproved by the Institutional Animal Care and Use Committee (IACUC) atBoston University Medical Center (#14388).

Transmission and Scanning Electron Microscopy

For transmission electron microscopy, kidneys were dissected from Robo2homozygous null mice and podocyte specific knockout mice, fixed in PLPat 4° C. overnight, and then incubated in 2% glutaraldehyde in 0.15 Msodium cacodylate for 6 hours. After washing in 1×PBS, fixed kidneyswere dehydrated in graded ethanol, embedded in Epon, sectioned, andstained with uranyl acetate and lead citrate. Ultrathin kidney sectionswere prepared and examined using a JEM-1011 electron microscope.Wild-type littermates were used as controls. For scanning electronmicroscopy, kidney samples from Robo2 homozygous null mice, podocytespecific knockout mice, nephrin homozygous null mice, and Robo2-nephrindouble homozygous mice were prepared following the protocol describedpreviously (Friedman and Ellisman, 1981) with minor modifications.Briefly, the kidney was perfused with 2.5% glutaraldehyde and 2%paraformaldehyde solution in 0.1M cacodylate buffer (Karnovsky'sfixative, Electron Microscopy Sciences), and subsequently fixed in theKarnovsky's fixative for 24 hrs followed by postfixation in 2% osmiumtetraoxide solution (Electron Microscopy Sciences). Kidney samples werecryofractured, dehydrated and dried using hexamethyldisilazane (ElectronMicroscopy Sciences). Kidney samples were imaged using an Amray 1000Aand Jeol 6340F scanning electron microscopes. Three glomeruli from eachanimal were examined to provide representative images.

Mice Kidney Pathology Studies, Quantification of Podocyte Number, andProteinuria Analysis

For kidney pathological studies, kidneys were dissected and fixed in 4%paraformaldehyde overnight, and then treated with a graded ethanolseries for paraffin embedding. The kidney paraffin blocks were sectionedat 4 μm using a MT-920 microtome (MICROM) and stained using standardPeriodic acid-Schiff (PAS) or eosin hematoxylin (H&E) methods. Theglomeruli were examined and assessed for matrix deposition, segmentalglomerulosclerosis, and dilatations of the Bowman's space using anOlympus BHT light microscope equipped with a SPOT digital camera system.For quantification of podocyte number, WT1 was used as a podocytenuclear marker and immunoperoxidase staining was performed on kidneysections following the protocol described previously (Sanden et al.,2003). Briefly, paraffin embedded kidney sections from 4 one-year oldRobo2^(del5/flox); Tg^(Nphs2-Cre+) podocyte-specific knockout mice and 4age-matched wild-type control mice were sectioned at 4 μm and stainedwith WT1 antibody (Santa Cruz Biotechnology) after microwave antigenretrieval. Biotinylated secondary antibody and Vectastain ABC kit(Vector Laboratories) were used to detect WT1 signal. WT1 positivepodocyte nuclei in each glomerular cross section were counted in total165 glomeruli from four mutant mice and 166 glomeruli from four controlmice. For proteinuria analysis, “spot” urine specimens from 6 weeks oldmice were examined using a sensitive murine albuminuria ELISAquantitation kit (Exocell) according to manufacturer's instruction andurine dipstick (Multistix from Bayer, Ind.) as a screening method. Urinealbumin was normalized with creatinine to provide an albumin/creatinineratio. Creatinine in urine was determined using the creatinine detectionkit (Sigma) according to manufacturer's instruction. Urine albumin wasalso examined by 12% SDS-PAGE and blotted with anti-albumin antibody (MPBiomedicals). The data from mutants and controls were analyzed usingone-way ANOVA, Student t-test, and Chi-square test.

TABLE 1 Quantitative Analysis of Glomeruli with Increased MatrixExpansion in 2 to 9 Months Old Robo2 Podocyte Specific Knockout Mice(Mutant) Compared to Controls (Wild type) Glomeruli with % of GlomeruliTotal mesangial with mesangial Mouse Age glomeruli matrix matrixGenotype ID# (months) counted expansion expansion* Mutant 4048 2 96 1312.35% Mutant 1721 3 107 18 16.82% Mutant 4005 6 103 17 16.51% Mutant1190 7 102 20 19.61% Mutant 2396 9 80 14 17.50% Mutant 488 82 16.80%total Wild type 4058 2 90 4 4.44% Wild type 4052 3 105 6 5.71% Wild type3919 6 107 4 3.74% Wild type 1191 7 103 5 4.85% Wild type 2385 9 106 54.72% Wild type 511 24 4.70% total *p < 0.01, n = 5, t-test.

TABLE 2 Quantitative Analysis of Glomeruli with Increased MatrixExpansion in 12-months-old Robo2 Podocyte-Specific Knockout Mice(Mutant) Compared to Controls (Wild type) Glomeruli with % of GlomeruliTotal mesangial with mesangial Mouse Age glomeruli matrix matrixGenotype ID# (months) counted expansion expansion* Mutant 1844 12 136 1712.50% Mutant 1847 12 125 18 14.40% Mutant 1877 12 127 11 8.66% Mutant1878 12 132 20 15.15% Mutant 1948 12 142 28 19.72% Mutant 662 94 14.20%total Wild type 1901 12 125 5 4.00% Wild type 2429 12 179 8 4.47% Wildtype 2834 12 154 9 5.84% Wild type 2836 12 159 7 4.40% Wild type 2837 12124 5 4.03% Wild type 741 34 4.59% total *p < 0.01, n = 5, t-test.

TABLE 3 Morphology Analysis of Glomeruli with Dilated Bowman's Space inNphs1^(−/−) Single-Homozygous (Robo2^(+/−); Nphs1^(−/−)) Compared toRobo2^(−/−); Nphs1^(−/−) Double-Homozygous Newborn Mice by HistologyTotal Glomeruli % of Glomeruli glomeruli with dilated with dilatedGenotype counted Bowman's space Bowman's space Robo2^(+/−); Nphs1^(−/−)122 31 25.4% Robo2^(−/−); Nphs1^(−/−) 55 2 3.6% Robo2^(−/−); Nphs1^(+/−)158 3 1.9% Robo2^(+/+); Nphs1^(+/+) 271 1 0.4% Note: The glomerulus wasscored as positive with dilated Bowman's space if the glomerulusdisplayed similar phenotype as shown in FIG. S4U was observed, and wasscored as negative if similar glomerulus as shown in FIG. 8V-8X wasobserved. Three mice from each genotype were analyzed. Robo2^(−/−)single homozygous (Robo2^(−/−); Nphs1^(+/−)) and wild-type (Robo2^(+/+);Nphs1^(+/+)) were used as controls.

TABLE 4 Morphology Analysis of Glomerular Podocyte Interdigitating FootProcess (FP) Phenotype in Nphs1^(−/−) Single-Homozygous (Robo2^(+/−);Nphs1^(−/−)) Compared to Robo2^(−/−); Nphs1^(−/−) Double-HomozygousNewborn Mice by Scanning Electron Microscopy Total Glomeruli with % ofGlomeruli with glomeruli interdigitating interdigitating FP Genotypecounted FP structure structure Robo2^(+/−); Nphs1^(−/−) 15 1 6.67% Robo2^(−/−); Nphs1^(−/−) 16 12  75% Robo2^(−/−); Nphs1^(+/−) 13 13 100%Robo2^(+/+); Nphs1^(+/+) 13 13 100%

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We claim:
 1. A pharmaceutical composition comprising a Roundabout 2(ROBO2) inhibitor that inhibits ROBO2 biological activity, wherein theROBO2 inhibitor is a soluble ROBO2 protein that (i) comprisesimmunoglobulin (Ig) motifs 1 and 2 of said ROBO2, (ii) does not compriseIg motifs 3, 4, and 5 of said ROBO2, and (iii) does not comprisefibronectin type III (FNIII) repeats of said ROBO2; and apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein the ROBO2 inhibitor prevents or reduces binding ofthe ROBO2 to SLIT2, to Nck, or to both.
 3. The pharmaceuticalcomposition of claim 1, wherein said soluble ROBO2 protein binds to SLITwith a binding affinity K_(D) (dissociation constant) value of 10⁻⁸ M orless.
 4. The pharmaceutical composition of claim 1, wherein the ROBO2inhibitor is a dominant negative soluble ROBO2 protein that reduces thebinding of ROBO2 to SLIT by at least 30%, relative to binding of ROBO2to SLIT in the absence of said soluble ROBO2 protein.
 5. Thepharmaceutical composition of claim 4, wherein the ROBO2 inhibitor is afusion polypeptide comprising the dominant negative soluble ROBO2protein.
 6. The pharmaceutical composition of claim 1, wherein thesoluble ROBO2 protein comprises amino acid residues 30-129 and 135-221of SEQ ID NO:
 3. 7. The pharmaceutical composition of claim 1, whereinsaid soluble ROBO2 protein does not comprise the intracellular domain ofsaid ROBO2.
 8. The pharmaceutical composition of claim 7, wherein saidintracellular domain comprises amino acid residues 881-1378 of SEQ IDNO:
 3. 9. The pharmaceutical composition of claim 1, wherein said Igmotif 1 comprises amino acid residues 30-129 of SEQ ID NO:
 3. 10. Thepharmaceutical composition of claim 1, wherein said Ig motif 2 comprisesamino acid residues 135-221 of SEQ ID NO:
 3. 11. The pharmaceuticalcomposition of claim 1, wherein said Ig motif 1 comprises amino acidresidues 30-129 of SEQ ID NO: 3, and said Ig motif 2 comprises aminoacid residues 135-221 of SEQ ID NO: 3.